ANTI-Aß OLIGOMER HUMANIZED ANTIBODY

ABSTRACT

An anti-Aβ oligomer humanized antibody which does not bind to Aβ monomers and specifically binds only to Aβ oligomers; an anti-cognitive dysfunction agent, an agent for treating Alzheimer&#39;s disease, an agent for suppressing formation of neuritic plaque and an inhibitor of formation of Aβ amyloid fiber comprising the antibody as an active ingredient; a method for at least one of preventing and treating cognitive dysfunction or Alzheimer&#39;s disease, comprising the step of administering the antibody; and a method for suppressing progression of Alzheimer&#39;s disease, comprising the step of administering the antibody.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an antibody which specifically binds toan amyloid-β (hereinafter, also referred to as Aβ) protein oligomer anduse thereof.

2. Brief Description of the Background Art

It is considered based on various pieces of evidence that the decline inmemory in Alzheimer's disease (hereinafter, referred to as AD) is causedas a result of synapse dysfunction due to soluble oligomers of amyloid-βprotein (hereinafter, amyloid-β protein is also referred to as Aβ, andamyloid-β protein oligomer is also referred to as Aβ oligomer)(Non-Patent Documents 1 and 2).

Accordingly, there is a possibility that excessive accumulation ordeposition of Aβ oligomers in a brain triggers a series of pathologicalcascades which may cause AD. This indicates a possibility that medicaltreatment targeting Aβ oligomers may be effective in delaying orpreventing the onset and the progression in the disease stage of AD.

However, the knowledge relating to the neurodegeneration caused by amolecule responsible for this amyloid-cascade hypothesis as its corefactor, particularly by Aβ oligomers was mainly demonstrated inexperiments in vitro (Non-Patent Document 3) and was not directlydemonstrated in vivo.

Since the structures specific to Aβ oligomers have not been studied inthe in vivo experiments which have been reported before (Non-PatentDocument 4), synaptic toxicity due to endogenous Aβ oligomers has notbeen clarified.

In addition, although studies have been carried out in various AD modelmice, neuronal toxicity of Aβ oligomers in the brain of an AD patienthas not yet been revealed.

Furthermore, it has not yet been established why the formation ofneurofibrillary tangle (hereinafter, simply referred to as NFT) and theloss of nerve cell occurs prior to pathogenesis of neuritic plaque inthe human entorhinal cortex and how Aβ oligomers relate to these tissuedegeneration and dysfunctions.

As antibodies against Aβ oligomers, anti-Aβ oligomer mouse monoclonalantibody NAB61 (Non-Patent Document 4), 1A9, 2C3, E12, 1C10, and 4D3(Patent Document 1) are known.

It is known that generally, when a non-human antibody such as a mouseantibody is administered to human, it is recognized as a foreignsubstance so that a human antibody for mouse antibody (human anti mouseantibody: HAMA) is induced in the human body. It is known that HAMAreacts with the administered mouse antibody to thereby induce sideeffects (Non-patent Documents 5 to 8), quickens disappearance of themouse antibody from the body (Non-patent Documents 6, 9 and 10) anddecreases therapeutic effect of the mouse antibody (Non-patent Documents11 and 12).

In order to solve these problems, attempts have been made to prepare arecombinant antibody such as a human chimeric antibody and a humanizedantibody from a non-human antibody using genetic recombinationtechniques.

A human chimeric antibody, a humanized antibody and the like havevarious advantages in clinidal application to human in comparison with anon-human antibody such as a mouse antibody. For example, it has beenreported that its immunogenicity was decreased and its blood half-lifewas prolonged in a test using monkey, compared to a mouse antibody(Non-patent Document 13).

That is, since a human chimeric antibody, a humanized antibody and thelike cause fewer side effects in human than non-human antibodies, it isexpected that its therapeutic effect is sustained for a prolonged time.

Also, since a recombinant antibody such as a human chimeric antibody, ahumanized antibody and a human antibody is prepared using recombinationtechniques, it can be prepared as various forms of molecules.

For example, γ1 subclass can be used as a heavy chain (hereinafterreferred to as “H chain”) constant region (hereinafter referred to as “Cregion”) of a human antibody (H chain C region is referred to as “CH”)to produce a recombinant antibody having high effector functions such asantibody-dependent cellular cytotoxicity (hereinafter referred to as“ADCC activity”). In addition, γ2 or γ4 subclass can be used as a heavychain to produce a recombinant antibody which has decreased effectorfunction and is expected to prolong of its blood half life in comparisonwith mouse antibodies (Non-patent Document 14).

Particularly, since cytotoxic activities such as complement-dependentcytotoxicity (hereinafter referred to as “CDC activity”) and ADCCactivity via the Fc region (the region after the antibody heavy chainhinge region) of an antibody are important for the therapeutic efficacy,a human chimeric antibody, a humanized antibody or a human antibody ispreferred compared to a non-human animal antibody such a mouse antibody(Non-patent Documents 15 and 16).

In addition, with recent advance in protein engineering and geneticengineering, the recombinant antibody can also be prepared as anantibody fragment having small molecular weight, such as Fab, Fab′,F(ab′)₂, a single chain antibody (hereinafter referred to as “scFv”)(Non-patent Document 17), a dimerized V region fragment (hereinafterreferred to as “Diabody”) (Non-patent Document 18), a disulfidestabilized V region fragment (hereinafter referred to as “dsFv”)(Non-patent Document 19), and a peptide comprising a CDR (Non-patentDocument 20). These antibody fragments have a greater advantage intransfer to target tissues than whole antibody molecules (Non-patentDocument 21).

The above-mentioned facts indicate that a human chimeric antibody, ahumanized antibody, a human antibody or an antibody fragment thereof ismore preferable as the antibody to be used for the clinical applicationto human than non-human antibodies, such as a mouse antibody.

CITATION LIST Patent Literature

Patent Literature 1: WO2009/051220

Non-Patent Literature

Non-patent Literature 1: Klein W L, Trends Neurosci 24:219-224, 2001.

Non-patent Literature 2: Selkoe D J, Science 298:789-791, 2002.

Non-patent Literature 3: Hass C et al, Nature Review 8:101-12, 2007.

Non-patent Literature 4: Lee E B, et al, J. Biol. Chem. 281:4292-4299,2006.

Non-patent Literature 5: J. Clin. Oncol., 2,881 (1984)

Non-patent Literature 6: Blood, 65, 1349 (1985)

Non-patent Literature 7: J. Natl. Cancer Inst., 80, 932 (1988)

Non-patent Literature 8: Proc. Natl. Acad. Sci., USA, 82, 1242 (1985)

Non-patent Literature 9: J. Nucl. Med, 26, 1011 (1985)

Non-patent Literature 10: J. Natl. Cancer Inst., 80, 937 (1988)

Non-patent Literature 11: J. Immunol., 135, 1530 (1985)

Non-patent Literature 12: Cancer Res., 46, 6489 (1986)

Non-patent Literature 13: Cancer Res., 56, 1118 (1996)

Non-patent Literature 14: Immunol., 85, 668 (1995)

Non-patent Literature 15: J. Immunol, 144, 1382 (1990)

Non-patent Literature 16: Nature, 322, 323 (1988)

Non-patent Literature 17: Science,242, 423 (1988)

Non-patent Literature 18: Nature Biotechnol., 15, 629 (1997)

Non-patent Literature 19: Molecular Immunol., 32, 249 (1995)

Non-patent Literature 20: J. Biol. Chem., 271, 2966 (1996)

Non-patent Literature 21: Cancer Res., 52, 3402 (1992)

SUMMARY OF THE INVENTION

The above Non-Patent Document 4 and Patent Document 1 discloseantibodies against Aβ oligomers. However, these antibodies bind not onlyto an Aβ oligomer but also to an Aβ monomer. Therefore, there is aconcern about central side-effects of the antibody during the antibodytherapy for AD, which targets the pathology in the brain.

For this reason, the purpose of the present invention is to provide ahumanized antibody which does not bind to an Aβ monomer and specificallybinds only to an Aβ oligomer and the use thereof. More specifically, thepurpose is to provide an antibody which specifically binds to an Aβoligomer, a method for measuring an Aβ oligomer using the antibody, amethod for diagnosing AD using the antibody, and a medicament comprisingthe antibody.

As a result of intensive deliberation in view of the above problem, thepresent inventors have found a humanized antibody which does not bind toan Aβ monomer and specifically binds only to an Aβ oligomer andaccomplished the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an amino acid sequence of H chain variable region HV0 andamino acid residues modified from the amino acid sequence represented bySEQ ID NO:12 in HV2, HV3,HV4, HV5, HV7a, HV7b, HV9 and HV 12. The firstand second lines in the drawing indicate the amino acid numbers in the Hchain variable region, and the letters in the other respective linesindicate the substituted amino acid (represented by single letter code).

FIG. 2 shows an amino acid sequence of L chain variable region LV0 andamino acid residues modified from the amino acid sequence represented bySEQ ID NO:14 in LV2a, LV2b, LV2c, LV2d, LV3 and LV4. The first andsecond rows in the drawing indicate the amino acid numbers in the Lchain variable region, and the letters in the respective rows indicatethe altered amino acid (represented by one letter).

FIG. 3 shows sensorgrams obtained by measuring the binding activities ofeach anti-Aβ oligomer humanized antibody to Aβ monomers using a Biacoresystem.

DETAILED DESCRIPTION OF THE INVENTION

That is, the present invention relates to the following 1 to 9.

-   1. A humanized antibody which does not bind to an Aβ protein monomer    and binds to an Aβ oligomer, and which comprises the following (a)    antibody heavy chain variable region and (b) antibody light chain    variable region:-   (a) an antibody heavy chain variable region comprising the amino    acid sequence represented by SEQ ID NO:12 or an amino acid sequence    in which at least one modification among amino acid modifications    for substituting Lys at position 12 with Val, Val at position 20    with Leu, Arg at position 38 with Lys, Gln at position 39 with Leu,    Leu at position 45 with Phe, Met at position 48 with Ile, Arg at    position 67 with Lys, Val at position 68 with Ala, Ile at position    70 with Leu, Ala at position 72 with Val, Thr at position 74 with    Lys and Ala at position 97 with Thr is carried out in the amino acid    sequence represented by SEQ ID NO:12,-   (b) an antibody light chain variable region comprising the amino    acid sequence represented by SEQ ID NO:14 or an amino acid sequence    in which at least one modifications among amino acid modifications    for substituting Ile at position 2 with Val, Pro at position 15 with    Leu, Gln at position 50 with Lys, and Tyr at position 92 with Phe is    carried out in the amino acid sequence represented by SEQ ID NO:14.-   2. A humanized antibody according to the above 1, wherein the    antibody heavy chain variable region comprises the amino acid    sequence represented by SEQ ID NO:12 or 15 and the antibody light    chain variable region comprises the amino acid sequence represented    by SEQ ID NO:14, 16, or 17.-   3. An anti-cognitive dysfunction agent, comprising the humanized    antibody according to the above 1 or 2 as an active ingredient.-   4. A medicament for treating Alzheimer's disease, comprising the    humanized antibody according to the above 1 or 2 as an active    ingredient.-   5. An agent for suppressing formation of neuritic plaque, comprising    the humanized antibody according to the above 1 or 2 as an active    ingredient.-   6. An inhibitor of formation of Aβ amyloid fiber, comprising the    humanized antibody according to the above 1 or 2 as an active    ingredient.-   7. A method for at least one of prevention and treatment of    cognitive dysfunction, comprising administering the humanized    antibody according to the above 1 or 2.-   8. A method for at least one of prevention and treatment of    Alzheimer's disease, comprising administering the humanized antibody    according to the above 1 or 2.-   9. A Method for suppressing progression of Alzheimer's disease,    comprising administering the humanized antibody according to the    above 1 or 2.

The antibody of the present invention is expected to establish methodsfor prevention and treatment of AD and a diagnostic marker at an earlystage by targeting an Aβ protein which is a causative molecule of AD.

There is a concern about intracerebral transfer of the antibody duringthe antibody therapy for AD which targets the pathology in the brain.However, there is a possibility that the antibody of the presentinvention is applicable for the clinical therapy by administration to aperipheral vein, and it can be considered that development in theantibody treatment of AD should be accelerated at once.

The anti-Aβ oligomer humanized antibody of the present invention(hereinafter, also referred to as the antibody of the present inventionor the humanized antibody of the present invention) is a humanizedantibody characterized in that it binds to an Aβ oligomer and does notbind to an Aβ monomer. The antibody of the present invention ispreferably an isolated antibody, a purified antibody, or an antibodycomposition.

The isolated antibody, the purified antibody, and the antibodycomposition are the antibodies which substantially comprise 100% of thedesired antibody and do not contain any impurities such as contaminantproteins in the production of the antibody, derived fromantibody-producing cells, tissues, antibody-producing animals and thelike.

The antibody is a heterotetrameric protein constituted by two heavychains (H chains) and two light chains (L chains). The antibody isclassified as a polyclonal antibody and a monoclonal antibody whichrecognized a single antigen.

The polyclonal antibody is a mixture of the antibodies which recognize asingle antigen. Examples of the polyclonal antibody include antiserum ofa host animal immunized with an antigen.

The monoclonal antibody is an antibody secreted by a single clone ofantibody-producing cells, and recognizes only one epitope (also calledantigen determinant) and has the uniformity in amino acid sequence(primary structure).

The antibody of the present invention is preferably a monoclonalantibody. Examples of the monoclonal antibody in the present inventioninclude a monoclonal antibody in which complementarity determiningregions (hereinafter, referred to as CDRs) 1 to 3 of the antibody heavychain (hereinafter, referred to as an H chain) comprise the amino acidsequences represented by SEQ ID NOs:1 to 3, respectively, and the CDRs 1to 3 in the L chain comprise the amino acid sequences represented by SEQID NOs:4 to 6, respectively, a monoclonal antibody in which the H chainvariable region (hereinafter, referred to as VH) of the antibodycomprises the amino acid sequence represented by SEQ ID NO:8 and the Lchain variable region (hereinafter, referred to as VL) comprises theamino acid sequence represented by SEQ ID NO:10, and an anti-Aβ oligomermouse monoclonal antibody 6E4.

As an epitope, a single amino acid sequence which is recognized andbound by the monoclonal antibody, a conformation comprising the aboveamino acid sequence, the above amino acid sequence to which a sugarchain binds, and a conformation comprising the above amino acidsequences to which sugar chains bind can be exemplified.

The epitope to be recognized by the antibody of the present inventionmay be any protein so long as it includes at least one of an Aβ proteinand the fragment thereof, and it exists on an Aβ oligomer which forms acomplex.

Examples of the epitope to which the antibody of the present inventionbinds include an epitope comprising a primary amino acid sequence of Aβwhich is exposed on an Aβ oligomer, an epitope comprising a conformationof an Aβ oligomer and the like.

It is known that the Aβ protein as a main component of amyloid is apeptide comprising 40 to 42 amino acids and is produced from a precursorprotein called an amyloid precursor protein (hereinafter, referred to asAPP) by the action of protease.

Amyloid molecules produced from APP include a non-fibrillar-polymer ofsoluble monomers and soluble oligomers in addition to the amyloid fibercollected in an ultracentrifugal sediment fraction.

In the present invention, the Aβ oligomer is a non-fibrillar-polymer,and is an Aβ oligomer which comprises at least one of an Aβ protein anda fragment thereof and forms a complex.

Specifically, examples of an Aβ oligomer include an Aβ40(Aβ1-40)oligomer, an Aβ42(Aβ1-42) oligomer, and an Aβ oligomer comprising atleast one of Aβ40 and Aβ42.

In addition, examples of the Aβ oligomer in the present invention alsoinclude an Aβ oligomer comprising an Aβ fragment with the loss of theN-terminal of Aβ in at least one of Aβ40 and Aβ42.

Specifically, the Aβ42 oligomer in the present invention are moleculeshaving a molecular weight of 45 to 160 kDa measured by SDS-PAGE and amolecular weight of 22.5 to 1,035 kDa measured by Blue Native-PAGE.

The Aβ42 oligomer is recovered mainly through >100 kDa retaining liquidin the molecular sieve. In addition, the Aβ oligomer shows a mixture ofconfiguration which consists of particle-shaped molecules, beadedmolecules, and circular molecules with the heights of 1.5 to 3.1 nmunder an atomic force microscope.

Moreover, the above Aβ42 oligomer is eluted to a void volume fraction 8of the molecular weight 680 kDa or more and to a fraction 15 of themolecular weight 17 to 44 kDa boundary by gel filteration method.

The antibody of the present invention can be used as long as it is ahumanized antibody which binds to an Aβ oligomer and does not bind to anAβ monomer, and the derivation and shape thereof are not limited.

It is preferable that the antibody of the present invention does notrecognize a soluble amyloid β (Aβ) monomer which is a physiologicalmolecule and reacts only with a soluble Aβ oligomer.

In the present invention, binding only to a soluble Aβ oligomer withoutbinding to a soluble Aβ monomer means that, among Aβ monomers and Aβoligomers which are separated by ultrafiltration and a molecular sieve,the antibody does not recognaize monomers (about 4.5 kDa), butspecifically recognize a soluble Aβ oligomer which is equal to or largerthan Aβ dimmers. Accordingly, it is preferable that the antibody of thepresent invention specifically binds to a soluble Aβ oligomer which isequal to, or larger than an Aβ dimer.

The antibody of the present invention has preferably at least oneactivity among the following (1) to (5):

-   (1) anti-neurotoxicity activity;-   (2) Aβ amyloid fiber formation suppressing activity;-   (3) specificity for recognizing only an Aβ oligomer;-   (4) ability of capturing an Aβ oligomer in AD brains-   (5) ability of preventing AD-like pathogenesis (decline in memory,    Aβ accumulation level in brains) in an APPswe-transgenic mouse    (Tg2576).

The activities of the antibody of the present invention relating to theabove (1) to (5) can be confirmed using the method disclosed in WO2009/051220.

The antibody of the present invention can be prepared as a recombinantantibody. In the present invention, the recombinant antibody includesantibodies produced by recombination technology such as a human chimericantibody, a humanized antibody (or a human CDR-grafted antibody), ahuman antibody and an antibody fragment thereof.

The recombinant antibodiey, having a character of monoclonal antibodies,low immunogenecity and prolonged half-life in blood is preferable as atherapeutic agent. Examples of the recombinant antibody include anantibody derived by modification of the monoclonal antibody of thepresent invention using recombination technology.

The human chimeric antibody is an antibody comprising a heavy chainvariable region (hereinafter referred to as “VH”) and a light chainvariable region (hereinafter referred to as “VL”) of an antibody of anon-human animal and a heavy chain constant region (hereinafter referredto as “CH”) and a light chain constant region (hereinafter referred toas “CL”) of a human antibody.

The human chimeric antibody can be produced by obtaining cDNAs encodingthe above VH and VL from a hybridoma which produces a monoclonalantibody that specifically binds to an Aβ oligomer, inserting each ofthem into an expression vector for animal cell comprising DNAs encodingthe CH and CL to thereby construct a vector for expression of humanchimeric antibody, and then introducing the vector into an animal cellto express the antibody.

As the above CH of the human chimeric antibody, any CH can be used, solong as it belongs to human immunoglobulin (hereinafter referred to as“hIg”), and those belonging to the hIgG class are preferred, and any oneof the subclasses belonging to the hIgG class, such as hIgG1, hIgG2,hIgG3 and hIgG4, can be used.

As the above CL of the human chimeric antibody, any CL can be used, solong as it belongs to the hIg class, and those belonging to κ class or λclass can be used.

The humanized antibody of the present invention is an antibody in whichamino acid sequences of CDRs of VH and VL of an antibody derived from anon-human animal are grafted into appropriate positions of VH and VL ofa human antibody.

The humanized antibody can be produced by designing amino acid sequenceof V regions in which the amino acid sequences of CDRs of both VH and VLof the monoclonal antibody which is produced by a non-human animalhybridoma and specifically binds to an Aβ oligomer are grafted intoframework regions (hereinafter referred to as “FR”) of VH and VL of anyhuman antibody, respectively, constructing cDNAs encoding the V regions,inserting each of them into a expression vector for animal cellscomprising genes encoding CH and CL of a human antibody to therebyconstruct a vector for expression of humanized antibody, and introducingit into an animal cell to thereby express and produce the humanizedantibody.

As the amino acid sequences of FRs of VH and VL of the humanizedantibody, any amino acid sequences can be used, so long as they areamino acid sequences of VH and VL, respectively, derived from a humanantibody.

Examples include amino acid sequences of FRs of VH and VL of humanantibodies registered in database such as Protein Data Bank, commonamino acid sequences of each subgroup of FRs of VH and VL of humanantibodies described in Sequences of Proteins of Immunological Interest,US Dept. Health and Human Services (1991) and the like.

Examples of the humanized antibody of the present invention include ahumanized antibody in which CDRs 1 to 3 of VH of the antibody comprisethe amino acid sequences represented by SEQ ID NOs:1 to 3, respectively,and CDRs 1 to 3 of VL of the antibody comprise the amino acid sequencesrepresented by SEQ ID NOs:4 to 6, respectively.

The humanized antibody of the present invention is preferably ahumanized antibody comprising at least one of the following (a) VH and(b) VL. In addition, in the following (a) and (b), the number of thesubstitutions to be introduced is not limited:

-   (a) VH comprising the amino acid sequence represented by SEQ ID    NO:12 or an amino acid sequence in which at least one amino acid    residue selected from Lys at position 12, Val at position 20, Arg at    position 38, Gln at position 39, Leu at position 45, Met at position    48, Arg at position 67, Val at position 68, Ile at position 70, Ala    at position 72, Thr at position 74 and Ala at position 97 in the    amino acid sequence represented by SEQ ID NO:12 is substituted with    another amino acid residue;-   (b) VL comprising the amino acid sequence represented by SEQ ID    NO:14 or an amino acid sequence in which at least one amino acid    residue selected from Ile at position 2, Pro at position 15, Gln at    position 50, and Tyr at position 92 in the amino acid sequence    represented by SEQ ID NO:14 is substituted with another amino acid    residue.

Preferable examples of VH contained in the humanized antibody of thepresent invention include, for example, VH comprising an amino acidsequence in which Lys at position 12, Val at position 20, Arg atposition 38, Gln at position 39, Leu at position 45, Met at position 48,Arg at position 67, Val at position 68, Ile at position 70, Ala atposition 72, Thr at position 74 and Ala at position 97 in the amino acidsequence represented by SEQ ID NO:12 are substituted with other aminoacid residues.

In addition, VH selected from the following (1) to (9) is alsopreferable as VH contained in the humanized antibody of the presentinvention:

-   (1) VH comprising an amino acid sequence in which Lys at position    12, Val at position 20, Arg at position 38, Gln at position 39, Leu    at position 45, Met at position 48, Arg at position 67, Val at    position 68, Ala at position 72, Thr at position 74, and Ala at    position 97 in the amino acid sequence represented by SEQ ID NO:12    are substituted with other amino acid residues;-   (2) VH comprising an amino acid sequence in which Lys at position    12, Arg at position 38, Gln at position 39, Leu at position 45, Met    at position 48, Arg at position 67, Val at position 68, Ala at    position 72, Thr at position 74 and Ala at position 97 in the amino    acid sequence represented by SEQ ID NO:12 are substituted with other    amino acid residues;-   (3) VH comprising an amino acid sequence in which Lys at position    12, Gln at position 39, Leu at position 45, Met at position 48, Arg    at position 67, Val at position 68, Ala at position 72, Thr at    position 74 and Ala at position 97 in the amino acid sequence    represented by SEQ ID NO:12 are substituted with other amino acid    residues;-   (4) VH comprising an amino acid sequence in which Gln at position    39, Leu at position 45, Met at position 48, Arg at position 67, Val    at position 68, Thr at position 74 and Ala at position 97 in the    amino acid sequence represented by SEQ ID NO:12 are substituted with    other amino acid residues;-   (5) VH comprising an amino acid sequence in which Leu at position    45, Met at position 48, Arg at position 67, Val at position 68, Ala    at position 72, Thr at position 74 and Ala at position 97 in the    amino acid sequence represented by SEQ ID NO:12 are substituted with    other amino acid residues;-   (6) VH comprising an amino acid sequence in which Gln at position    39, Leu at position 45, Met at position 48, Arg at position 67 and    Ala at position 97 in the amino acid sequence represented by SEQ ID    NO:12 are substituted with other amino acid residues;-   (7) VH comprising an amino acid sequence in which Leu at position    45, Met at position 48, Arg at position 67 and Ala at position 97 in    the amino acid sequence represented by SEQ ID NO:12 are substituted    with other amino acid residues;-   (8) VH comprising an amino acid sequence in which Leu at position    45, Met at position 48 and Ala at position 97 in the amino acid    sequence represented by SEQ ID NO:12 are substituted with other    amino acid residues;-   (9) VH comprising an amino acid sequence in which Leu at position 45    and Ala at position 97 in the amino acid sequence represented by SEQ    ID NO:12 are substituted with other amino acid residues.

Examples of the amino acid sequence of VH include an amino acid sequencein which at least one modification selected from modifications ofsubstituting Lys at position 12 with Val, Val at position 20 with Leu,Arg at position 38 with Lys, Gln at position 39 with Leu, Leu atposition 45 with Phe, Met at position 48 with Ile, Arg at position 67with Lys, Val at position 68 with Ala, Ile at position 70 with Leu, Alaat position 72 with Val, Thr at position 74 with Lys and Ala at position97 with Thr in the amino acid sequence represented by SEQ ID NO:12 isintroduced.

More specific examples of the amino acid sequence of VH include theamino acid sequences in which the following twelve to one substitutionsare introduced.

Specific examples of the amino acid sequence of VH in which twelvesubstitutions are introduced include an amino acid sequence in which Lysat position 12 is substituted with Val, Val at position 20 issubstituted with Leu, Arg at position 38 is substituted with Lys, Gln atposition 39 is substituted with Leu, Leu at position 45 is substitutedwith Phe, Met at position 48 is substituted with Ile, Arg at position 67is substituted with Lys, Val at position 68 is substituted with Ala, Ileat position 70 is substituted with Leu, Ala at position 72 issubstituted with Val, Thr at position 74 is substituted with Lys and Alaat position 97 is substituted with Thr in the amino acid sequencerepresented by SEQ ID NO:12.

Specific examples of the amino acid sequence of VH in which elevensubstitutions are introduced include the following (1) to (12) aminoacid sequences:

-   (1) an amino acid sequence in which Val at position 20 is    substituted with Leu, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (2) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (3) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (4) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted With Ala, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (5) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (6) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (7) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (8) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (9) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (10) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Ile at position 70 is substituted with Leu, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (11) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Ile at position 70 is substituted with Leu, Ala at position 72 is    substituted with Val and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (12) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Ile at position 70 is substituted with Leu, Ala at position 72 is    substituted with Val and Thr at position 74 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12.

Specific examples of the amino acid sequence of VH in which tensubstitutions are introduced include the following (1) to (66) aminoacid sequences, for example:

-   (1) an amino acid sequence in which Arg at position 38 is    substituted with Lys, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (2) an amino acid sequence in which Val at position 20 is    substituted with Leu, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (3) an amino acid sequence in which Val at position 20 is    substituted with Leu, Arg at position 38 is substituted with Lys,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (4) an amino acid sequence in which Val at position 20 is    substituted with Leu, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (5) an amino acid sequence in which Val at position 20 is    substituted with Leu, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (6) an amino acid sequence in which Val at position 20 is    substituted with Leu, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Val at position 68 is substituted with Ala, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (7) an amino acid sequence in which Val at position 20 is    substituted with Leu, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (8) an amino acid sequence in which Val at position 20 is    substituted with Leu, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (9) an amino acid sequence in which Val at position 20 is    substituted with Leu, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (10) an amino acid sequence in which Val at position 20 is    substituted with Leu, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (11) an amino acid sequence in which Val at position 20 is    substituted with Leu, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val and Thr at position 74 is    substituted with Lys in the amino acid sequence represented by SEQ    ID NO: 12;-   (12) an amino acid sequence in which Lys at position 12 is    substituted with Val, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (13) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (14) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (15) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (16) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Val at position 68 is substituted with Ala, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (17) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (18) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (19) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (20) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (21) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val and Thr at position 74 is    substituted with Lys in the amino acid sequence represented by SEQ    ID NO:12;-   (22) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (23) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Gln at position 39 is substituted with Leu, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (24) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (25) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Val at position 68 is substituted with Ala, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (26) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (27) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (28) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, He at position 70 is substituted with Leu, Thr    at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (29) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (30) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val and Thr at position 74 is    substituted with Lys in the amino acid sequence represented by SEQ    Ill NO:12;-   (31) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (32) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Leu at position 45 is    substituted with Phe, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (33) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Val at position 68 is substituted with Ala, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (34) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (35) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (36) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (37) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (38) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val and Thr at position 74 is    substituted with Lys in the amino acid sequence represented by SEQ    ID NO:12;-   (39) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (40) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Met at position 48 is substituted with Ile,    Vat at position 68 is substituted with Ala, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (41) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (42) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (43) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (44) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, He at position 70 is substituted with Leu, Ala    at position 72 is substituted with Val and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (45) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Vat at position 68 is    substituted with Ala, He at position 70 is substituted with Leu, Ala    at position 72 is substituted with Val and Thr at position 74 is    substituted with Lys in the amino acid sequence represented by SEQ    ID NO:12;-   (46) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Val at position 68 is substituted with Ala, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted With Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (47) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Arg at position 67 is substituted with Lys, Be at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (48) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (49) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (50) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (51) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val and Thr at position 74 is    substituted with Lys in the amino acid sequence represented by SEQ    ID NO:12;-   (52) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with He, Ile at position 70 is    substituted with Leu, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (53) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Val at position 68 is    substituted with Ala, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (54) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (55) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (56) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Val at position 68 is    substituted with Ala, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val and Thr at position 74 is    substituted with Lys in the amino acid sequence represented by SEQ    ID NO:12;-   (57) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (58) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Ile at position 70 is substituted with Leu,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (59) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (60) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val and Thr at position 74 is    substituted with Lys in the amino acid sequence represented by SEQ    ID NO:12;-   (61) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (62) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Ala at position 72 is substituted with Val and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (63) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Ala at position 72 is substituted with Val and Thr at position 74 is    substituted with Lys in the amino acid sequence represented by SEQ    ID NO:12;-   (64) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with He, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Ile at position 70 is substituted with Leu and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (65) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Ile at position 70 is substituted with Leu and Thr at position 74 is    substituted with Lys in the amino acid sequence represented by SEQ    ID NO:12;-   (66) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Arg at position 38 is substituted with Lys, Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Ile at position 70 is substituted with Leu and Ala at position 72 is    substituted with Val in the amino acid sequence represented by SEQ    ID NO:12.

Specific examples of the amino acid sequence of VH in which ninesubstitutions are introduced include the following (1) to (10) aminoacid sequences:

-   (1) an amino acid sequence in which Arg at position 38 is    substituted with Lys, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ala at position 72 is    substituted with Val, Thr at position 74 is substituted with Lys and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (2) an amino acid sequence in which Lys at position 12 is    substituted with Val, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ala at position 72 is    substituted with Val, Thr at position 74 is substituted with Lys and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (3) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ala at position 72 is    substituted with Val, Thr at position 74 is substituted with Lys and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (4) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ala at position 72 is    substituted with Val, Thr at position 74 is substituted with Lys and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (5) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ala at position 72 is    substituted with Val, Thr at position 74 is substituted with Lys and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (6) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Val at position 68 is substituted with Ala, Ala at position 72 is    substituted with Val, Thr at position 74 is substituted with Lys and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (7) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Ala at position 72 is    substituted with Val, Thr at position 74 is substituted with Lys and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (8) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Thr at position 74 is substituted with Lys and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (9) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ala at position 72 is substituted with Val and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (10) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ala at position 72 is substituted with Val and    Thr at position 74 is substituted with Lys in the amino acid    sequence represented by SEQ ID NO:12.

Specific examples of the amino acid sequence of VH in which eightsubstitutions are introduced include the following (1) to (47) aminoacid sequences:

-   (1) an amino acid sequence in which Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (2) an amino acid sequence in which Arg at position 38 is    substituted with Lys, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (3) an amino acid sequence in which Arg at position 38 is    substituted with Lys, Gln at position 39 is substituted with. Leu,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (4) an amino acid sequence in which Arg at position 38 is    substituted with Lys, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (5) an amino acid sequence in which Arg at position 38 is    substituted with Lys, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Val at position 68 is substituted with Ala,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (6) an amino acid sequence in which Arg at position 38 is    substituted with Lys, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Ala at position 72 is substituted with Vat, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (7) an amino acid sequence in which Arg at position 38 is    substituted with Lys, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (8) an amino acid sequence in which Arg at position 38 is    substituted with Lys, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with He, Arg at position 67 is substituted with Lys, Val    at position 68 is substituted with Ala, Ala at position 72 is    substituted with Val and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (9) an amino acid sequence in which Arg at position 38 is    substituted with Lys, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ala at position 72 is    substituted with Val and Thr at position 74 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (10) an amino acid sequence in which Lys at position 12 is    substituted with Val, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (11) an amino acid sequence in which Lys at position 12 is    substituted with Val, Gln at position 39 is substituted with Leu,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (12) an amino acid sequence in which Lys at position 12 is    substituted with Val, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (13) an amino acid sequence in which Lys at position 12 is    substituted with Val, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Val at position 68 is substituted with Ala,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (14) an amino acid sequence in which Lys at position 12 is    substituted with Val, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (15) an amino acid sequence in which Lys at position 12 is    substituted with Val, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (16) an amino acid sequence in which Lys at position 12 is    substituted with Val, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ala at position 72 is    substituted with Val and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (17) an amino acid sequence in which Lys at position 12 is    substituted with Val, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ala at position 72 is    substituted with Val and Thr at position 74 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (18) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (19) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (20) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Leu at position 45 is substituted with Phe, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (21) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Val at position 68 is substituted with Ala,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (22) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (23) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (24) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ala at position 72 is    substituted with Val and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (25) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with He, Arg at position 67 is substituted with Lys, Val    at position 68 is substituted with Ala, Ala at position 72 is    substituted with Val and Thr at position 74 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (26) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ Ill NO:12;-   (27) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (28) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Met at position 48 is    substituted with Ile, Val at position 68 is substituted with Ala,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (29) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (30) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (31) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ala at position 72 is    substituted with Val and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (32) an amino acid sequence in Which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ala at position 72 is    substituted with Val and Thr at position 74 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (33) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Val at position 68 is substituted with Ala,    Ala at position 72 is substituted with Vat, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (34) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Arg at position 67 is substituted with Lys,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (35) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (36) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ala at position 72 is    substituted with Val and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (37) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala, Ala at position 72 is    substituted with Val and Thr at position 74 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (38) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Ala at position 72 is substituted with Val, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (39) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Val at position 68 is substituted with Ala, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (40) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Val at position 68 is substituted with Ala, Ala at position 72 is    substituted with Val and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (41) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Val at position 68 is substituted with Ala, Ala at position 72 is    substituted with Val and Thr at position 74 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (42) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (43) an amino acid sequence in which Lys at position' 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Ala at position 72 is    substituted with Val and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (44) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Ala at position 72 is    substituted with Val and Thr at position 74 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (45) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (46) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala and Thr at position 74 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (47) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala and Ala at position 72 is substituted with Val    in the amino acid sequence represented by SEQ ID NO:12.

Specific examples of the amino acid sequence of VH in which sevensubstitutions are introduced include the following (1) to (22) aminoacid sequences:

-   (1) an amino acid sequence in which Lys at position 12 is    substituted with Val, Val at position 20 is substituted with Leu,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (2) an amino acid sequence in which Lys at position 12 is    substituted with Val, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (3) an amino acid sequence in which Lys at position 12 is    substituted with Val, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (4) an amino acid sequence in which Lys at position 12 is    substituted with Val, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Ile at position 70 is substituted with Leu and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (5) an amino acid sequence in which Lys at position 12 is    substituted with Val, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Ala at position 72 is substituted with Val and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (6) an amino acid sequence in which Lys at position 12 is    substituted with Val, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (7) an amino acid sequence in which Val at position 20 is    substituted with Leu, Arg at position 38 is substituted with Lys,    Gln at position 39 is substituted with Leu, Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (8) an amino acid sequence in which Val at position 20 is    substituted with Leu, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (9) an amino acid sequence in which Val at position 20 is    substituted with Leu, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Ile at position 70 is substituted with Leu and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (10) an amino acid sequence in which Val at position 20 is    substituted with Leu, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Ala at position 72 is substituted with Val and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (11) an amino acid sequence in which Val at position 20 is    substituted with Leu, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (12) an amino acid sequence in which Arg at position 38 is    substituted with Lys, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Val at position 68 is substituted with Ala and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (13) an amino acid sequence in which Arg at position 38 is    substituted with Lys, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Ile at position 70 is substituted with Leu and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (14) an amino acid sequence in which Arg at position 38 is    substituted with Lys, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Ala at position 72 is substituted with Val and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (15) an amino acid sequence in which Arg at position 38 is    substituted with Lys, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (16) an amino acid sequence in which Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Ile at position 70 is substituted with Leu and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (17) an amino acid sequence in which Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Ala at position 72 is substituted with Val and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (18) an amino acid sequence in which Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (19) an amino acid sequence in which Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Ile at position 70 is substituted with Leu,    Ala at position 72 is substituted with Val and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (20) an amino acid sequence in which Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Ile at position 70 is substituted with Leu,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (21) an amino acid sequence in which Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (22) an amino acid sequence in which Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Ala at position 72 is substituted with Val,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12.

Specific examples of the amino acid sequence of VH in which sixsubstitutions are introduced include the following (1) to (7) amino acidsequences:

-   (1) an amino acid sequence in which Lys at position 12 is    substituted with Val, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (2) an amino acid sequence in which Val at position 20 is    substituted with Leu, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (3) an amino acid sequence in which Arg at position 38 is    substituted with Lys, Gln at position 39 is substituted with Leu,    Leu at position 45 is substituted with Phe, Met at position 48 is    substituted with Ile, Arg at position 67 is substituted with Lys and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (4) an amino acid sequence in which Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Val at position 68 is substituted with Ala and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (5) an amino acid sequence in which Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Ile at position 70 is substituted with Leu and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (6) an amino acid sequence in which Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Ala at position 72 is substituted with Val and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (7) an amino acid sequence in which Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys, Thr at position 74 is substituted with Lys and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12.

Specific examples of the amino acid sequence of VH in which fivesubstitutions are introduced include the following (1) to (8) amino acidsequences:

-   (1) an amino acid sequence in which Lys at position 12 is    substituted with Val, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (2) an amino acid sequence in which Val at position 20 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (3) an amino acid sequence in which Arg at position 38 is    substituted with Lys, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (4) an amino acid sequence in which Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile, Arg at position 67 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (5) an amino acid sequence in which Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Val at position 68 is    substituted with Ala and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (6) an amino acid sequence in which Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Ile at position 70 is    substituted with Leu and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (7) an amino acid sequence in which Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Ala at position 72 is    substituted with Val and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (8) an amino acid sequence in which Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys, Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12.

Specific examples of the amino acid sequence of VH in which foursubstitutions are introduced include the following (1) to (9) amino acidsequences:

-   (1) an amino acid sequence in which Lys at position 12 is    substituted with Val, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (2) an amino acid sequence in which Val at position 20 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (3) an amino acid sequence in which Arg at position 38 is    substituted with Lys, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    NO:12;-   (4) an amino acid sequence in which Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe,    Met at position 48 is substituted with Ile and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (5) an amino acid sequence in which Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Arg at position 67 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (6) an amino acid sequence in which Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Val at position 68 is substituted with Ala and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (7) an amino acid sequence in which Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Ile at position 70 is substituted with Leu and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (8) an amino acid sequence in which Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Ala at position 72 is substituted with Val and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12;-   (9) an amino acid sequence in which Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile,    Thr at position 74 is substituted with Lys and Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12.

Specific examples of the amino acid sequence of VH in which threesubstitutions are introduced include the following (1) to (19) aminoacid sequences, for example:

-   (1) an amino acid sequence in which Lys at position 12 is    substituted with Val, Leu at position 45 is substituted with Phe and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (2) an amino acid sequence in which Val at position 20 is    substituted with Leu, Leu at position 45 is substituted with Phe and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (3) an amino acid sequence in which Arg at position 38 is    substituted with Lys, Leu at position 45 is substituted with Phe and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (4) an amino acid sequence in which Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (5) an amino acid sequence in which Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (6) an amino acid sequence in which Leu at position 45 is    substituted with Phe, Arg at position 67 is substituted with Lys and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (7) an amino acid sequence in which Leu at position 45 is    substituted with Phe, Val at position 68 is substituted with Ala and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (8) an amino acid sequence in which Leu at position 45 is    substituted with Phe, Ile at position 70 is substituted with Leu and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (9) an amino acid sequence in which Leu at position 45 is    substituted with Phe, Ala at position 72 is substituted with Val and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (10) an amino acid sequence in which Leu at position 45 is    substituted with Phe, Thr at position 74 is substituted with Lys and    Ala at position 97 is substituted with Thr in the amino acid    sequence represented by SEQ ID NO:12;-   (11) an amino acid sequence in which Lys at position 12 is    substituted with Val, Leu at position 45 is substituted with Phe and    Met at position 48 is substituted with Ile in the amino acid    sequence represented by SEQ ID NO:12;-   (12) an amino acid sequence in which Val at position 20 is    substituted with Leu, Leu at position 45 is substituted with Phe and    Met at position 48 is substituted with Ile in the amino acid    sequence represented by SEQ ID NO:12;-   (13) an amino acid sequence in which Arg at position 38 is    substituted with Lys, Leu at position 45 is substituted with Phe and    Met at position 48 is substituted with He in the amino acid sequence    represented by SEQ ID NO:12;-   (14) an amino acid sequence in which Gln at position 39 is    substituted with Leu, Leu at position 45 is substituted with Phe and    Met at position 48 is substituted with Ile in the amino acid    sequence represented by SEQ ID NO:12;-   (15) an amino acid sequence in which Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile and    Arg at position 67 is substituted with Lys in the amino acid    sequence represented by SEQ ID NO:12;-   (16) an amino acid sequence in which Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile and    Val at position 68 is substituted with Ala in the amino acid    sequence represented by SEQ ID NO:12;-   (17) an amino acid sequence in which Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile and    Ile at position 70 is substituted with Leu in the amino acid    sequence represented by SEQ ID NO:12;-   (18) an amino acid sequence in which Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile and    Ala at position 72 is substituted with Val in the amino acid    sequence represented by SEQ ID NO:12;-   (19) an amino acid sequence in which Leu at position 45 is    substituted with Phe, Met at position 48 is substituted with Ile and    Thr at position 74 is substituted with Lys in the amino acid    sequence represented by SEQ ID NO:12.

Specific examples of the amino acid sequence of VH in which twosubstitutions are introduced include the following (1) to (66) aminoacid sequences, for example:

-   (1) an amino acid sequence in which Lys at position 12 is    substituted with Val and Val at position 20 is substituted with Leu    in the amino acid sequence represented by SEQ ID NO:12;-   (2) an amino acid sequence in which Lys at position 12 is    substituted with Val and Arg at position 38 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (3) an amino acid sequence in which Lys at position 12 is    substituted with Val and Gln at position 39 is substituted with Leu    in the amino acid sequence represented by SEQ 1D NO:12;-   (4) an amino acid sequence in which Lys at position 12 is    substituted with Val and Leu at position 45 is substituted with Phe    in the amino acid sequence represented by SEQ ID NO:12;-   (5) an amino acid sequence in which Lys at position 12 is    substituted with Val and Met at position 48 is substituted with Ile    in the amino acid sequence represented by SEQ ID NO:12;-   (6) an amino acid sequence in which Lys at position 12 is    substituted with Val and Arg at position 67 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (7) an amino acid sequence in which Lys at position 12 is    substituted with Val and Val at position 68 is substituted with Ala    in the amino acid sequence represented by SEQ ID NO:12;-   (8) an amino acid sequence in which Lys at position 12 is    substituted with Val and Ile at position 70 is substituted with Leu    in the amino acid sequence represented by SEQ ID NO:12;-   (9) an amino acid sequence in which Lys at position 12 is    substituted with Val and Ala at position 72 is substituted with Val    in the amino acid sequence represented by SEQ ID NO:12;-   (10) an amino acid sequence in which Lys at position 12 is    substituted with Val and Thr at position 74 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (11) an amino acid sequence in which Lys at position 12 is    substituted with Val and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (12) an amino acid sequence in which Val at position 20 is    substituted with Leu and Arg at position 38 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (13) an amino acid sequence in which Val at position 20 is    substituted with Leu and Gln at position 39 is substituted with Leu    in the amino acid sequence represented by SEQ ID NO:12;-   (14) an amino acid sequence in which Val at position 20 is    substituted with Leu and Leu at position 45 is substituted with Phe    in the amino acid sequence represented by SEQ ID NO:12;-   (15) an amino acid sequence in which Val at position 20 is    substituted with Leu and Met at position 48 is substituted with Ile    in the amino acid sequence represented by SEQ ID NO:12;-   (16) an amino acid sequence in which Val at position 20 is    substituted with Leu and Arg at position 67 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (17) an amino acid sequence in which Val at position 20 is    substituted with Leu and Val at position 68 is substituted with Ala    in the amino acid sequence represented by SEQ ID NO:12;-   (18) an amino acid sequence in which Val at position 20 is    substituted with Leu and Ile at position 70 is substituted with Leu    in the amino acid sequence represented by SEQ ID NO:12;-   (19) an amino acid sequence in which Val at position 20 is    substituted with Leu and Ala at position 72 is substituted with Val    in the amino acid sequence represented by SEQ ID NO:12;-   (20) an amino acid sequence in which Val at position 20 is    substituted with Leu and Thr at position 74 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (21) an amino acid sequence in which Val at position 20 is    substituted with Leu and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (22) an amino acid sequence in which Arg at position 38 is    substituted with Lys and Gln at position 39 is substituted with Leu    in the amino acid sequence represented by SEQ ID NO:12;-   (23) an amino acid sequence in which Arg at position 38 is    substituted with Lys and Leu at position 45 is substituted with Phe    in the amino acid sequence represented by SEQ ID NO:12;-   (24) an amino acid sequence in which Arg at position 38 is    substituted with Lys and Met at position 48 is substituted with Ile    in the amino acid sequence represented by SEQ ID NO:12;-   (25) an amino acid sequence in which Arg at position 38 is    substituted with Lys and Arg at position 67 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (26) an amino acid sequence in which Arg at position 38 is    substituted with Lys and Val at position 68 is substituted with Ala    in the amino acid sequence represented by SEQ ID NO:12;-   (27) an amino acid sequence in which Arg at position 38 is    substituted with Lys and Ile at position 70 is substituted with Leu    in the amino acid sequence represented by SEQ ID NO:12;-   (28) an amino acid sequence in which Arg at position 38 is    substituted with Lys and Ala at position 72 is substituted with Val    in the amino acid sequence represented by SEQ ID NO:12;-   (29) an amino acid sequence in which Arg at position 38 is    substituted with Lys and Thr at position 74 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (30) an amino acid sequence in which Arg at position 38 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (31) an amino acid sequence in which Gln at position 39 is    substituted with Leu and Leu at position 45 is substituted with Phe    in the amino acid sequence represented by SEQ ID NO:12;-   (32) an amino acid sequence in which Gln at position 39 is    substituted with Leu and Met at position 48 is substituted with Ile    in the amino acid sequence represented by SEQ ID NO:12;-   (33) an amino acid sequence in which Gln at position 39 is    substituted with Leu and Arg at position 67 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (34) an amino acid sequence in which Gln at position 39 is    substituted with Leu and Val at position 68 is substituted with Ala    in the amino acid sequence represented by SEQ ID NO:12;-   (35) an amino acid sequence in which Gln at position 39 is    substituted with Leu and Ile at position 70 is substituted with Leu    in the amino acid sequence represented by SEQ ID NO:12;-   (36) an amino acid sequence in which Gln at position 39 is    substituted with Leu and Ala at position 72 is substituted with Val    in the amino acid sequence represented by SEQ ID NO:12;-   (37) an amino acid sequence in which Gln at position 39 is    substituted with Leu and Thr at position 74 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (38) an amino acid sequence in which Gln at position 39 is    substituted with Leu and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (39) an amino acid sequence in which Leu at position 45 is    substituted with Phe and Met at position 48 is substituted with Ile    in the amino acid sequence represented by SEQ ID NO:12;-   (40) an amino acid sequence in which Leu at position 45 is    substituted with Phe and Arg at position 67 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (41) an amino acid sequence in which Leu at position 45 is    substituted with Phe and Val at position 68 is substituted with Ala    in the amino acid sequence represented by SEQ ID NO:12;-   (42) an amino acid sequence in which Leu at position 45 is    substituted with Phe and Ile at position 70 is substituted with Leu    in the amino acid sequence represented by SEQ ID NO:12;-   (43) an amino acid sequence in which Leu at position 45 is    substituted with Phe and Ala at position 72 is substituted with Val    in the amino acid sequence represented by SEQ ID NO:12;-   (44) an amino acid sequence in which Leu at position 45 is    substituted with Phe and Thr at position 74 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (45) an amino acid sequence in which Leu at position 45 is    substituted with Phe and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (46) an amino acid sequence in which Met at position 48 is    substituted with Ile and Arg at position 67 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (47) an amino acid sequence in which Met at position 48 is    substituted with Ile and Val at position 68 is substituted with Ala    in the amino acid sequence represented by SEQ ID NO:12;-   (48) an amino acid sequence in which Met at position 48 is    substituted with Ile and Ile at position 70 is substituted with Leu    in the amino acid sequence represented by SEQ ID NO:12;-   (49) an amino acid sequence in which Met at position 48 is    substituted with Ile and Ala at position 72 is substituted with Val    in the amino acid sequence represented by SEQ ID NO:12;-   (50) an amino acid sequence in which Met at position 48 is    substituted with Ile and Thr at position 74 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (51) an amino acid sequence in which Met at position 48 is    substituted with Ile and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (52) an amino acid sequence in which Arg at position 67 is    substituted with Lys and Val at position 68 is substituted with Ala    in the amino acid sequence represented by SEQ ID NO:12;-   (53) an amino acid sequence in which Arg at position 67 is    substituted with Lys and Ile at position 70 is substituted with Leu    in the amino acid sequence represented by SEQ ID NO:12;-   (54) an amino acid sequence in which Arg at position 67 is    substituted with Lys and Ala at position 72 is substituted with Val    in the amino acid sequence represented by SEQ ID NO:12;-   (55) an amino acid sequence in which Arg at position 67 is    substituted with Lys and Thr at position 74 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (56) an amino acid sequence in which Arg at position 67 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (57) an amino acid sequence in which Val at position 68 is    substituted with Ala and Ile at position 70 is substituted with Leu    in the amino acid sequence represented by SEQ ID NO:12;-   (58) an amino acid sequence in which Val at position 68 is    substituted with Ala and Ala at position 72 is substituted with Val    in the amino acid sequence represented by SEQ ID NO:12;-   (59) an amino acid sequence in which Val at position 68 is    substituted with Ala and Thr at position 74 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (60) an amino acid sequence in which Val at position 68 is    substituted with Ala and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (61) an amino acid sequence in which Ile at position 70 is    substituted with Leu and Ala at position 72 is substituted with Val    in the amino acid sequence represented by SEQ ID NO:12;-   (62) an amino acid sequence in which Ile at position 70 is    substituted with Leu and Thr at position 74 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (63) an amino acid sequence in which Ile at position 70 is    substituted with Leu and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (64) an amino acid sequence in which Ala at position 72 is    substituted with Val and Thr at position 74 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:12;-   (65) an amino acid sequence in which Ala at position 72 is    substituted with Val and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12;-   (66) an amino acid sequence in which Thr at position 74 is    substituted with Lys and Ala at position 97 is substituted with Thr    in the amino acid sequence represented by SEQ ID NO:12.

Specific examples of the amino acid sequence of VH in which asubstitution is introduced include the following (1) to (12) amino acidsequences, for example:

-   (1) an amino acid sequence in which Lys at position 12 is    substituted with Val in the amino acid sequence represented by SEQ    ID NO:12;-   (2) an amino acid sequence in which Val at position 20 is    substituted with Leu in the amino acid sequence represented by SEQ    ID NO:12;-   (3) an amino acid sequence in which Arg at position 38 is    substituted with Lys in the amino acid sequence represented by SEQ    ID NO:12;-   (4) an amino acid sequence in which Gln at position 39 is    substituted with Leu in the amino acid sequence represented by SEQ    ID NO:12;-   (5) an amino acid sequence in which Leu at position 45 is    substituted with Phe in the amino acid sequence represented by SEQ    ID NO:12;-   (6) an amino acid sequence in which Met at position 48 is    substituted with Ile in the amino acid sequence represented by SEQ    ID NO:12;-   (7) an amino acid sequence in which Arg at position 67 is    substituted with Lys in the amino acid sequence represented by SEQ    ID NO:12;-   (8) an amino acid sequence in which Val at position 68 is    substituted with Ala in the amino acid sequence represented by SEQ    ID NO:12;-   (9) an amino acid sequence in which Ile at position 70 is    substituted with Leu in the amino acid sequence represented by SEQ    ID NO:12;-   (10) an amino acid sequence in which Ala at position 72 is    substituted with Val in the amino acid sequence represented by SEQ    ID NO:12;-   (11) an amino acid sequence in which Thr at position 74 is    substituted with Lys in the amino acid sequence represented by SEQ    ID NO:12;-   (12) an amino acid sequence in which Ala at position 97 is    substituted with Thr in the amino acid sequence represented by SEQ    ID NO:12.

Among the above amino acid sequences of VH, the amino acid sequencerepresented by SEQ ID NO:12, an amino acid sequence in which Lys atposition 12 is substituted with Val, Val at position 20 is substitutedwith Leu, Arg at position 38 is substituted with Lys, Gln at position 39is substituted with Leu, Leu at position 45 is substituted with Phe, Metat position 48 is substituted with Ile, Arg at position 67 issubstituted with Lys, Val at position 68 is substituted with Ala, Ile atposition 70 is substituted with Leu, Ala at position 72 is substitutedwith Val, Thr at position 74 is substituted with Lys and Ala at position97 is substituted with Thr in the amino acid sequence represented by SEQID NO:12 are preferable.

The humanized antibody of the present invention preferably includes VLcomprising an amino acid sequence in which Ile at position 2, Pro atposition 15, Gln at position 50, Tyr at position 92 in the amino acidsequence represented by SEQ ID NO:14 are substituted with other aminoacid residues, and preferable examples of VL of the present inventionalso include VL comprising an amino acid sequence in which Pro atposition 15, Gln at position 50, Tyr at position 92 in the amino acidsequence represented by SEQ ID NO:14 are substituted with other aminoacid residues.

In addition, VL selected from the following (1) to (4) is alsopreferable as VL contained in the humanized antibody of the presentinvention:

-   (1) VL comprising an amino acid sequence in which Gln at position 50    and Tyr at position 92 in the amino acid sequence represented by SEQ    ID NO:14 are substituted with other amino acid residues;-   (2) VI, comprising an amino acid sequence in which He at position 2    and Tyr at position 92 in the amino acid sequence represented by SEQ    ID NO:14 are substituted with other amino acid residues;-   (3) VL comprising an amino acid sequence in which Pro at position 15    and Tyr at position 92 in the amino acid sequence represented by SEQ    ID NO:14 are substituted with other amino acid residues;-   (4) VL comprising an amino acid sequence in which Ile at position 2    and Gln at position 50 in the amino acid sequence represented by SEQ    ID NO:14 is substituted with another amino acid residue.

The amino acid sequence of the VL include, for example, an amino acidsequence in which at least one modification selected from modificationsof substituting Ile at position 2 with Val, Pro at position 15 with Leu,Gln at position 50 with Lys and Tyr at position 92 with Phe in the aminoacid sequence represented by SEQ ID NO:14 is introduced.

More specific examples of the amino acid sequence of the VI, in theantibody of the present invention in which the above modifications areintroduced include, for example, the amino acid sequence of the VL inwhich the following four to one substitutions are introduced.

Specific examples of the amino acid sequence of VL in which foursubstitutions are introduced include an amino acid sequence in which Ileat position 2 is substituted with Val, Pro at position 15 is substitutedwith Leu, Gln at position 50 is substituted with Lys and Tyr at position92 is substituted with Phe in the amino acid sequence represented by SEQID NO:14.

Specific examples of the amino acid sequence of VL in which threesubstitutions are introduced include the following (1) to (4) amino acidsequences:

-   (1) an amino acid sequence in which Ile at position 2 is substituted    with Val, Pro at position 15 is substituted with Leu and Gln at    position 50 is substituted with Lys in the amino acid sequence    represented by SEQ ID NO:14;-   (2) an amino acid sequence in which Ile at position 2 is substituted    with Val, Pro at position 15 is substituted with Leu and Tyr at    position 92 is substituted with Phe in the amino acid sequence    represented by SEQ ID NO:14;-   (3) an amino acid sequence in which Ile at position 2 is substituted    with Val, Gln at position 50 is substituted with Lys and Tyr at    position 92 is substituted with Phe in the amino acid sequence    represented by SEQ ID NO:14;-   (4) an amino acid sequence in which Pro at position 15 is    substituted with Leu, Gln at position 50 is substituted with Lys and    Tyr at position 92 is substituted with Phe in the amino acid    sequence represented by SEQ ID NO:14.

Specific examples of the amino acid sequence of VL in which twosubstitutions arc introduced include the following (1) to (6) amino acidsequences:

-   (1) an amino acid sequence in which Ile at position 2 is substituted    with Val and Pro at position 15 is substituted with Leu in the amino    acid sequence represented by SEQ ID NO:14;-   (2) an amino acid sequence in which Ile at position 2 is substituted    with Val and Gln at position 50 is substituted with Lys in the amino    acid sequence represented by SEQ ID NO:14;-   (3) an amino acid sequence in which Ile at position 2 is substituted    with Val and Tyr at position 92 is substituted with Phe in the amino    acid sequence represented by SEQ ID NO:14;-   (4) an amino acid sequence in which Pro at position 15 is    substituted with Leu and Gln at position 50 is substituted with Lys    in the amino acid sequence represented by SEQ ID NO:14;-   (5) an amino acid sequence in which Pro at position 15 is    substituted with Leu and Tyr at position 92 is substituted with Phe    in the amino acid sequence represented by SEQ ID NO:14;-   (6) an amino acid sequence in which Gln at position 50 is    substituted with Lys and Tyr at position 92 is substituted with Phe    in the amino acid sequence represented by SEQ ID NO:14.

Specific examples of the amino acid sequence of VL in which onesubstitution is introduced include the following (1) to (4) amino acidsequences:

-   (1) an amino acid sequence in which Ile at position 2 is substituted    with Val in the amino acid sequence represented by SEQ ID NO:14;-   (2) an amino acid sequence in which Pro at position 15 is    substituted with Leu in the amino acid sequence represented by SEQ    ID NO:14;-   (3) an amino acid sequence in which Gln at position 50 is    substituted with Lys in the amino acid sequence represented by SEQ    ID NO:14;-   (4) an amino acid sequence in which Tyr at position 92 is    substituted with Phe in the amino acid sequence represented by SEQ    ID NO:14.

Among the above amino acid sequences of VL, the amino acid sequencerepresented by SEQ ID NO:14, an amino acid sequence in which Ile atposition 2 is substituted with Val, Pro at position 15 is substitutedwith Leu, Gln at position 50 is substituted with Lys and Tyr at position92 is substituted with Phe in the amino acid sequence represented by SEQID NO:14, and an amino acid sequence in which Gln at position 50 issubstituted with Lys and Tyr at position 92 is substituted with Phe inthe amino acid sequence represented by SEQ ID NO:14 are preferable.

Specific examples of the antibody of the present invention include anyone of the antibodies each of which is obtained by combining the abovementioned VH and VL, respectively.

Preferable examples of the antibody of the present invention include thefollowing antibodies (1) to (4), for example:

-   (1) a humanized antibody comprising VH which comprises the amino    acid sequence represented by SEQ ID NO:12 and VL which comprises the    amino acid sequence represented by SEQ ID NO:14;-   (2) a humanized antibody comprising VH which comprises the amino    acid sequence represented by SEQ ID NO:12 and VL which comprises any    one of the amino acid sequences shown in FIG. 2;-   (3) a humanized antibody comprising VH which comprises any one of    the amino acid sequences shown in FIG. 1 and VL which comprises the    amino acid sequence represented by SEQ ID NO:14;-   (4) a humanized antibody comprising VH which comprises any one of    the amino acid sequences shown in FIG. 1 and VL which comprises any    one of the amino acid sequences shown in FIG. 2.

Among the amino acid sequences of the VI-I shown in FIG. 1, 6E4HV0 andHV12 are preferable. In addition, among the amino acid sequences of theVL shown in FIG. 2, 6E4LV0, LV2a and LV4 are preferable.

Accordingly, as the humanized antibody comprising VH which comprises anyone of the amino acid sequences shown in FIG. 1 and VL which comprisesany one of the amino acid sequences shown in FIG. 2, the humanizedantibody comprising VH which comprises any one of the amino acidsequences among 6E4HV0 and HV12 shown in FIG. 1 and VL which comprisesany one of the amino acid sequences among 6E4LV0, LV2a and LV4 shown inFIG. 2 is preferable.

Specifically, examples include the humanized antibody comprising VHwhich comprises the amino acid sequences represented by SEQ ID NO:12 or15 and V L which comprises any one of the amino acid sequencesrepresented by SEQ ID NOs:14, 16 and 17.

As the combination of the amino acid sequence of VH shown in FIG. 1 andthe amino acid sequence of VL shown in FIG. 2, the combinations of HV0and LV0, HV0 and LV2a, HV0 and LV4 and HV12 and LV4 are preferable.

Accordingly, as the antibody of the present invention, the followinghumanized antibodies (1) to (4) are more preferable:

-   (1) a humanized antibody (HV0LV0) comprising VH which comprises the    amino acid sequence represented as HV0 in FIG. 1 and VL which    comprises the amino acid sequence represented as LV0 in FIG. 2;-   (2) a humanized antibody (HV0LV2a) comprising VH which comprises the    amino acid sequence represented as HV0 in FIG. 1 and VL which    comprises the amino acid sequence represented as LV2a in FIG. 2;-   (3) a humanized antibody (HV0LV4) comprising VH which comprises the    amino ‘acid sequence represented as HV0 in FIG. 1 and VL which    comprises the amino acid sequence represented as LV4 in FIG. 2;-   (4) a humanized antibody (HV12LV4) comprising VH which comprises the    amino acid sequence represented as HV12 in FIG. 1 and VL which    comprises the amino acid sequence represented as LV4 in FIG. 2.

Specific examples of the above antibodies include a humanized antibodyin which the combination of the amino acid sequences contained in VH andVL are SEQ ID NOs:12 and 14; SEQ ID NOs:12 and 16; SEQ ID NOs:12 and 17;and SEQ ID NOs:15 and 17.

In addition, examples of the humanized antibody of the present inventionincludes a humanized antibody which competes with the above humanizedantibody in the binding of an Aβ oligomer and a humanized antibody whichbinds to an epitope which is the same as the epitope recognized by theabove humanized antibody.

In the present invention, a human antibody is originally an antibodynaturally existing in the human body, and it also includes antibodiesobtained from a human antibody phage library, a human antibody-producingtransgenic animal, and the like, which are prepared based on the recentadvance in genetic engineering, cell engineering and developmentalengineering techniques.

The antibody existing in the human body can be prepared, for example byisolating a human peripheral blood lymphocyte, immortalizing it byinfecting with EB virus or the like and then cloning it to therebyobtain lymphocytes capable of producing the antibody, culturing thelymphocytes thus obtained, and purifying the antibody from thesupernatant of the culture.

The human antibody phage library is a library in which antibodyfragments such as Fab and scFv arc expressed on the phage surface byinserting a gene encoding an antibody prepared from a human B cell intoa phage gene.

A phage expressing an antibody fragment having the desired antigenbinding activity on the cell surface can be recovered from the library,using its activity to bind to an antigen-immobilized substrate as theindex. The antibody fragment can be converted further into a humanantibody molecule comprising two full H chains and two full L chains bygenetic engineering techniques.

A human antibody-producing transgenic animal is an animal in which ahuman antibody gene is integrated into cells. Specifically, a humanantibody-producing transgenic animal can be prepared by introducing agene encoding a human antibody into a mouse ES cell, grafting the EScell into an early stage embryo of other mouse and then developing itinto a complete animal.

A human antibody derived from the human antibody-producing transgenicnon-human animal can be prepared by obtaining a human antibody-producinghybridoma by a hybridoma preparation method usually carried out innon-human animals, culturing the obtained hybridoma and producing andaccumulating the human antibody in the supernatant of the culture.

An antibody or antibody fragment thereof in which one or more aminoacids are deleted, substituted, inserted or added into the amino acidsequence constituting the above antibody or antibody fragment, havingactivity similar to the above antibody or antibody fragment is alsoincluded in the antibody or antibody fragment of the present invention.

The number of amino acid residues which are deleted, substituted,inserted and/or added is one or more, and is not specifically limited,but it is within the range where deletion, substitution or addition ispossible by known methods such as the site-directed mutagenesisdescribed in Molecular Cloning, Second Edition, Cold Spring HarborLaboratory Press (1989); Current Protocols in Molecular Biology, JohnWilly & Sons (1987-1997); Nucleic Acids Research, 10, 6487 (1982), Proc.Natl. Acad. Sci. USA, 79, 6409 (1982); Gene, 34, 315 (1985), NucleicAcids Research, 13, 4431 (1985); Proc. Natl. Acad. Sci. USA, 82, 488(1985) or the like. For example, the number is 1 to dozens, preferably 1to 20, more preferably 1 to 10, and most preferably 1 to 5.

Deleting, substituting, inserting or adding one or more amino acidresidues in the amino acid sequence of the above antibody means thefollowings. That is, it means there is deletion, substitution, insertionor addition of one or plural amino acid residues at any positions in oneor plural amino acid sequences of the antibody within a single sequence.Also, the deletion, substitution, insertion or addition may occur at thesame time and the amino acid which is substituted, inserted or added maybe either a natural type or a non-natural type.

The natural type amino acid includes L-alanine, L-asparagine, L-asparticacid, L-glutamine, L: glutamic acid, glycine, L-histidine, L-isoleucine,L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine,L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine,L-cysteine and the like.

Preferable examples of mutually substitutable amino acids are shownbelow. The amino acids in the same group are mutually substitutable.

-   Group A: leucine, isoleucine, norleucine, valine, norvaline,    alanine, 2-aminobutanoic acid, methionine, O-methylserine,    t-butylglycine, t-butylalanine, cyclohexylalanine-   Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic    acid, 2-aminoadipic acid, 2-aminosuberic acid-   Group C: asparagine, glutamine-   Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid,    2,3-diaminopropionic acid-   Group E: proline, 3-hydroxyproline, 4-hydroxyproline-   Group F: serine, threonine, homoserine-   Group G: phenylalanine, tyrosine

The antibody fragment of the present invention includes Fab, F(ab′)₂,Fab′, scFv, diabody, dsFv, a peptide comprising CDR and the like.

An Fab is an antibody fragment having a molecular weight of about 50,000and having antigen binding activity, in which about a half of theN-terminal side of H chain and the entire L chain, among fragmentsobtained by treating an IgG antibody molecule with a protease, papain(cleaved at an amino acid residue at position 224 of the H chain), areboundlogether through a disulfide bond.

The Fab of the present invention can be obtained by treating amonoclonal antibody which specifically recognizes an Aβ oligomer andbinds to the extracellular domain, with a protease, papain. Also, theFab can be produced by inserting DNA encoding Fab of the antibody intoan expression vector for prokaryote or an expression vector foreukaryote, and introducing the vector into a prokaryote or eukaryote toexpress the Fab.

An F(ab′)₂ is an antibody fragment having a molecular weight of about100,000 and antigen binding activity and comprising two Fab regionswhich are bound in the hinge portion obtained by digesting the lowerpart of two disulfide bonds in the hinge region of IgG, with an enzyme,pepsin.

The F(ab′)₂ of the present invention can be obtained by treating amonoclonal antibody which specifically recognizes an Aβ oligomer andbinds to the extracellular domain, with a protease, pepsin. Also, theF(ab′)₂ can be produced by binding Fab′ described below via a thioetherbond or a disulfide bond.

An Fab′ is an antibody fragment having a molecular weight of about50,000 and antigen binding activity, which is obtained by cleaving adisulfide bond at the hinge region of the above F(ab′)₂.

The Fab′ of the present invention can be obtained by treating F(ab′)₂which specifically recognizes an Aβ oligomer and binds to theextracellular domain, with a reducing agent, dithiothreitol.

Also, the Fab′ can be produced by inserting DNA encoding Fab′ fragmentof the antibody into an expression vector for prokaryote or anexpression vector for eukaryote, and introducing the vector into aprokaryote or eukaryote to express the Fab′.

An scFv is a VH-P-VL or VL-P-VH polypeptide in which a VH chain and a VLchain are linked using an appropriate peptide linker (hereinafterreferred to as “P”) and is an antibody fragment having antigen bindingactivity.

The scFv of the present invention can be produced by obtaining cDNAsencoding VH and VL of a monoclonal antibody which specificallyrecognizes an Aβ oligomer and binds to the extracellular domain,constructing DNA encoding the scFv, inserting the DNA into an expressionvector for prokaryote or an expression vector for eukaryote, and thenintroducing the expression vector into a prokaryote or eukaryote toexpress the scFv.

A diabody is an antibody fragment wherein scFv is dimerized, and hasdivalent antigen binding activity. In the divalent antigen bindingactivity, two antigens may be the same or different.

The diabody of the present invention can be produced by obtaining cDNAsencoding VH and VL of a monoclonal antibody which specificallyrecognizes an Aβ oligomer and binds to the extracellular domain,constructing DNA encoding the scFv so that the length of the amino acidsequence of P is 8 or less residues, inserting the DNA into anexpression vector for prokaryote or an expression vector for eukaryote,and then introducing the expression vector into a prokaryote oreukaryote to express the diabody.

A dsFv is obtained by binding polypeptides in which one amino acidresidue of each of VH and VL is substituted with a cysteine residue viaa disulfide bond between the cysteine residues. The amino acid residueto be substituted with a cysteine residue can be selected based on athree-dimensional structure estimation of the antibody in accordancewith the method shown by Reiter et al. [Protein Engineering, 7, 697(1994)].

The dsFv of the present invention can be produced by obtaining cDNAsencoding VH and VL of a monoclonal antibody which specificallyrecognizes an Aβ oligomer and binds to the extracellular domain,constructing DNA encoding dsFv, inserting the DNA into an expressionvector for prokaryote or an expression vector for eukaryote, and thenintroducing the expression vector into a prokaryote or eukaryote toexpress the dsFv.

A peptide comprising CDR is constituted by including at least one regionor more of CDRs of VH or VL. A peptide comprising plural CDRs can beproduced by connecting CDRs directly or via an appropriate peptidelinker.

The peptide comprising CDR of the present invention can be produced byconstructing DNA encoding CDRs of VH and VL of a humanized antibodywhich specifically recognizes an Aβ oligomer of the present invention,inserting the DNA into an expression vector for prokaryote or anexpression vector for eukaryote, and then introducing the expressionvector into a prokaryote or eukaryote to express the peptide. Thepeptide comprising CDR can also be produced by a chemical synthesismethod such as Fmoc method and tBoc method.

The antibody of the present invention includes an antibody derivative inwhich the anti-Aβ oligomer humanized antibody of the present inventionor the antibody fragment thereof is chemically or genetically bound to aradioisotope, a low-molecular agent, a high-molecular agent, a protein,a therapeutic antibody and the like.

The antibody derivative of the present invention can be produced bychemically conjugating a radioisotope a low-molecular agent, ahigh-molecular agent, a protein, a therapeutic antibody and the like tothe N-terminal side or C-terminal side of an H chain or an L chain ofthe anti-Aβ oligomer humanized antibody or the antibody fragmentthereof, an appropriate substituent or side chain in the antibody or theantibody fragment, a sugar chain in the antibody or the antibodyfragment and the like [Antibody Engineering Handbook published by ChijinShokan (1994)].

Also, the antibody derivative of the present invention can begenetically produced by linking a DNA encoding the anti-Aβ oligomerhumanized antibody of the present invention or the antibody fragmentthereof to other DNA encoding a protein to be conjugated or DNA encodinga therapeutic antibody, inserting the DNA into a vector for expression,and introducing the expression vector into a host cell.

In the case where the above antibody derivative is used as a detectionregent, a regent for quantitative determination or a diagnostic agentfor the detection method, quantification method or diagnosis method,respectively, examples of the agent to which the anti-Aβ oligomerhumanized antibody or the antibody fragment thereof in the presentinvention is conjugated includes a label which is generally used inimmunological detecting or measuring method.

The label includes enzymes such as alkaline phosphatase, peroxidase andluciferase, luminescent materials such as acridinium ester and lophine,fluorescent materials such as fluorescein isothiocyanate (FITC) andtetramethyl rhodamine isothiocyanate (RITC).

Hereinafter, a method for producing the antibody is described.

1. Production of Anti-AP Oligomer Monoclonal Antibody

In the present invention, the anti-Aβ oligomer monoclonal antibody canbe produced in the following manner.

(1) Preparation of Antigen

As a method for producing an Aβ oligomer as antigens, a synthetic Aβ1-42(Peptide Institute, Inc. Osaka) is dissolved in deionized distilledwater or in 10 mmol/L of phosphate buffer and incubated at 37° C. for 18hours, and the peptide is separated by 4 to 12% SDS-PAGE, and theresulting material is visualized by CBB staining, only an Aβ1-42tetramer without contamination of an Aβ1-42 monomer are then collected,and thereby Aβ1-42 oligomers can be produced.

Aβ1-40 oligomers comprising more oligomers can be prepared by chemicalconjugation of 6-carboxytetramethylrhodamine (6-TAMRA) (SIGMA) to theN-terminal of the synthesized AP1-40 peptide using a conventionalmethod, followed by polymerization reaction with the synthetic Aβ1-40(Peptide Institute, Inc. Osaka).

(2) Immunization of Animals

First, 2.5 μg of Aβ1-42 tetramers or Aβ1-40 oligomers is emulsified bycomplete Freund's adjuvant. Then, the antigen is immunized on a footpadof a Balb-c mouse. And subsequently, six additional immunizations areperformed. The fusion between antibody-producing cells and myeloma cellscan be performed in a known method such as a method by Kohler andMilstein, et al. (Kohler. G and Milstein, C., Methods Enzymol. (1981)73, 3-46) or the like. Specifically, immunocytes producing the antibodyare obtained from inguinal lymph nodes of a mouse to which the antigenhas been immunized.

The immunization is carried out by administering the antigen to theanimal through subcutaneous, intravenous or intraperitoneal injectiontogether with an appropriate adjuvant such as complete Freund's adjuvantand combination of aluminum hydroxide gel with pertussis vaccine.

When a partial peptide is used as the antigen, a conjugate to a carrierprotein such as BSA (bovine serum albumin) and KLH (keyhole limpethemocyanin) is produced for use as the antigen.

The animal immunized with an antigen may be any animal, so long as ahybridoma can be prepared, and mouse, rat, hamster, chicken, rabbit orthe like is preferably used. Also, the antibody of the present inventionincludes an antibody produced by a hybridoma obtained by fusing amyeloma cell with the cell having antibody-producing activity derivedfrom such an animal after in vitro immunization.

(3) Preparation of Myeloma Cells

Established cell lines derived from mouse are used as myeloma cells.Examples include 8-azaguanine-resistant murine myeloma cell line(derived from BALB/c) P3-X63Ag8-U1 (P3-U1) [Current Topics inMicrobiology and Immunology, 18, 1 (1978)], P3-NS1/1-Ag41 (NS-1)[European J. Immunology, 6, 511 (1976)], SP2/0-Ag14 (SP-2) [Nature, 276,269 (1978)], P3-X63-Ag8653 (653) [J. Immunology, 123, 1548 (1979)],P3-X63-Ag8 (X63) [Nature, 256, 495 (1975)] and the like.

The myeloma cells are subcultured in a normal medium [RPMI1640 mediumcontaining glutamine, 2-mercaptoethanol, gentamicin, FBS and8-azaguanine]. The cells are transferred in the normal medium 3 or 4days before cell fusion to ensure the cell number of 2×10⁷ or more onthe day for fusion.

(4) Cell Fusion and Preparation of Monoclonal Antibody-ProducingHybridomas

A hybridoma can be produced by fusing the antibody-producing cells forfusion obtained in step (2) and the myeloma cells obtained in step (3)using polyethylene glycol 1500.

After the culturing, a portion of the culture supernatant is sampled andsubjected to hybridoma screening method such as dot-blotting and bindingassay to identify the cell population producing antibodies which isreactive to Aβ oligomers and not to Aβ monomers as described below.Then, cloning is carried out twice by a limiting dilution method [HTmedium (HAT medium without aminopterin) is used in the first round, andthe normal medium is used in the second round], and a hybridoma whichstably shows a high antibody titer is selected as the monoclonalantibody-producing hybridoma.

(5) Preparation of Purified Monoclonal Antibodies

The hybridoma cells producing a monoclonal antibody obtained by step (4)are administered by intraperitoneal injection into 8- to 10-week-oldmice or nude mice which is pretreated with pristane (0.5 mL of2,6,10,14-tetramethylpentadecane (pristane) is intraperitoneallyadministered, followed by feeding for 2 weeks).

The hybridoma forms ascites tumor in 10 to 21 days. The ascitic fluid iscollected from the mice, centrifuged to remove solids, subjected tosalting out with 40 to 50% ammonium sulfate and then precipitated bycaprylic acid, passed through a DEAE-Sepharose column, a protein Acolumn or a gel filtration column to collect IgG or IgM fractions aspurified monoclonal antibodies.

(6) Selection of Monoclonal Antibodies

As a method of screening the antibody, it is possible to exemplify amethod of selecting the antibody using a binding activity of theantibody to an Aβ oligomer as an index.

The binding activity of the antibody of the present invention againstthe antigen (Aβ oligomer) can be analyzed using at least one of themethods such as absorbance measurement method, enzyme linkedimmunosorbent assay method (dot blot method, ELISA), enzyme immunoassaymethod (EIA), radioimmunoassay method (RIA), immunofluorescence methodand Biacore (Biacore Life Science) using the surface plasmon resonancemethod (SPR method).

Specifically, the binding of the anti-AP oligomer monoclonal antibody tothe Aβ oligomer can be determined by the dot blot method in which 2.5 μlof Aβ31-42 (2.5 μg/dot) is pre-incubated for 18 hours, followed by beingimmobilized on a nitrocellulose membrane. After blocking thenon-specific binding site on the membrane with phosphate buffer solutioncontaining 5% of reduced-fat milk, 1% of BSA and 0.05% of Tween-20, themembrane is incubated with culture supernatant, and thereby the Aβoligomer binding antibody in the culture supernatant can be detected byhorseradish peroxidase labelled goat anti-mouse F(ab′)₂ (1:3000;Amersham) using an enhanced chemiluminescence (ECL) kit and LAS3000mini(Fujitsu, Tokyo, Japan) (WO 2009/051220).

The ELISA is carried out by immobilizing the antibody onto a plate,adding an antigen against the antibody onto the plate, and adding asample containing desirable antibodies such as the culture supernatantof the cells producing the antibody and the purified antibody. Next, asecondary antibody which recognizes the primary antibody and is taggedadded thereto by an enzyme such as alkaline phosphatase is addedthereto, and the plate is incubated. After washing, an enzyme substratesuch as p-nitrophenylphosphate is added to the plate and the absorbanceis measured to evaluate the antigen binding ability of the targetsample.

In addition, the reactivity of the antibody can also be determined byusing the sandwich enzyme linked immunosorbent assay usingchemiluminescence (chemiluminescence-ELISA) in order to specificallydetect Aβ oligomers and not to detect Aβ monomers in the presentinvention (WO 2009/051220).

In addition, in order to evaluate the effectiveness of the monoclonalantibody which has been administered to the periphery of a mouse invivo, HRP-labelled 6E10 (Senetek PLC, Napa, Calif., USA) human-specificoligomer ELISA can be carried out for Aβ oligomer analysis of plasma andorgan collected from the administered mouse in the present invention (WO2009/051220).

Moreover, it is possible to determined whether the anti-Aβ oligomerhumanized antibody of the present invention can bind to Aβ oligomers inAD brains by performing the immunoprecipitation experiment (Ghiso J, etal.: Biochem J, 1993) with an amyloid fraction (Matsubara E, et al.:Neurobiol Aging, 2004) containing a large amount of Aβ oligomers (WO2009/051220).

(7) Evaluation of Anti-Aβ Oligomer Antibody Activity

In the present invention, it is possible to determine that the anti-Aβoligomer antibody specifically binds to Aβ oligomers and has acytoprotective activity by Aβ incubation for Thioflavin (ThT) assay,Aβ-induced neurotoxicity assay, and measurement of the reactivityagainst Aβ oligomers with plural molecule sizes fractionated byultrafiltration and gel filtration, all of which arc disclosed inYamamoto N, et al.: J. Biol. Chem., 282:2646-2655, 2007 or WO2009/051220.

Furthermore, the Aβ amyloid fiber formation suppressing activity can bedetermined using ThT assay and electron microscope analysis.

In the present invention, the Aβ oligomer-trapping ability of both theantibodies in AD brains can be measured by determining the existence ofSDS-stable 4-, 5-, 8-, 12-mer in immunoprecipitation using the anti-Aβoligomer humanized antibody.

Moreover, in the present invention, examples of a method for determiningthe ability for the prophylaxis of the AD-like pathogenesis in anAPPswe-transgenic mouse (Tg2576) include a method in which the humanizedantibody of the present invention is administered to the mouse toexamine whether or not the AD-like pathogenesis such as memory disorder,neuritic plaque lesions, synapse dysfunction and Aβ accumulation can beprevented (WO 2009/051220).

2. Preparation of Recombinant Antibody

The method for producing a transformant to stably express a recombinantantibody and the recombinant antibody can be carried out as below.

(1) Construction of Vector for Expression of Recombinant Antibody

A vector for expression of recombinant antibody is an expression vectorfor animal cell into which DNAs encoding CH and CL of a human antibodyhave been inserted, and is constructed by cloning each of DNAs encodingCH and CL of a human antibody into an expression vector for animal cell.

The C region of a human antibody may be CH and CL of any human antibody.Examples include CH belonging to γ1 subclass, CL belonging to κ class,and the like. As the DNAs encoding CH and CL of a human antibody, achromosomal DNA comprising an exon and an intron or cDNA can be used.

As the expression vector for animal cell, any expression vector can beused, so long as a gene encoding the C region of a human antibody can beinserted thereinto and expressed therein. Examples include pAGE107[Cytotechnol., 3, 133 (1990)], pAGE103 [J. Biochem., 101, 1307 (1987)],pHSG274 [Gene, 27, 223 (1984)], pKCR [Proc. Natl. Acad. Sci. USA, 781527 (1981)], pSG1bd2-4 [Cytotechnol., 4, 173 (1990)], pSE1UK1Sed1-3[Cytotechnol., 13, 79 (1993)] and the like.

Examples of a promoter and enhancer used for an expression vector foranimal cell include an SV40 early promoter [J. Biochem., 101, 1307(1987)], a Moloney mouse leukemia virus LTR [Biochem. Biophys. Res.Commun., 149, 960 (1987)], an immunoglobulin H chain promoter [Cell, 41,479 (1985)] and enhancer [Cell, 33 717 (1983)] and the like.

The vector for expression of recombinant antibody may be either of atype in which a gene encoding an antibody H chain and a gene encoding anantibody L chain exist on separate vectors or of a type in which bothgenes exist on the same vector (tandem type). In respect of easiness ofconstruction of a vector for expression of recombinant antibody,easiness of introduction into animal cells, and balance between theexpression amounts of antibody H and L chains in animal cells, a tandemtype of the vector for expression of recombinant antibody is morepreferred [J. Immunol. Methods, 167, 271 (1994)].

Examples of the tandem type of the vector for expression of recombinantantibody include pKANTEX93 (WO 97/10354), pEE18 [Hybridoma, 17, 559(1998)], and the like.

(2) Preparation of cDNA Encoding V Region of Antibody Derived fromNon-Human Animal and Analysis of Amino Acid Sequence

mRNA is extracted from hybridoma cells producing an antibody derivedfrom a non-human animal to synthesize cDNA. The synthesized cDNA iscloned into a vector and the sequence analysis using a DNA sequencer iscarried out to determine the nucleotide sequences encoding VH and VL.

Whether the obtained cDNAs encode the full amino acid sequences of VLand VL of the antibody containing a secretory signal sequence can beconfirmed by estimating the full length of the amino acid sequences ofVH and VL from the determined nucleotide sequence and comparing themwith the full length of the amino acid sequences of VH and VL of knownantibodies [Sequences of Proteins of Immunological Interest, US Dept.Health and Human Services (1991)].

The length of the secretory signal sequence and N-terminal amino acidsequence can be deduced by comparing the full length of the amino acidsequences of VH and VL of the antibody comprising a secretory signalsequence with full length of the amino acid sequences of VH and VL ofknown antibodies [Sequences of Proteins of Immunological Interest, USDept. Health and Human Services (1991)], and the subgroup to which theybelong can also be known.

Furthermore, the amino acid sequence of each of CDRs of VH and VL can befound by comparing the obtained amino acid sequences with amino acidsequences of VH and VL of known antibodies [Sequences of Proteins ofImmunological Interest, US Dept. Health and Human Services (1991)].

(3) Construction of Vector for Expression of Human Chimeric Antibody

cDNAs encoding VH and VL of antibody of non-human animal are cloned inthe upstream of genes encoding CH or CL of human antibody of vector forexpression of recombinant antibody mentioned in the above (1) to therebyconstruct a vector for expression of human chimeric antibody.

For example, cDNAs encoding VH and VL are designed and constructed tocomprise the linker sequences encoding appropriate amino acids andhaving appropriate recognition sites of restriction enzyme so as toproduce a connection between the 3′-end of a cDNA of VH or VL derivedfrom a non-human animal antibody and the 5′-end of CH or CL derived froma human antibody.

The resultant each cDNA encoding VH and VL is cloned so that each ofthem is expressed in an appropriate form in the upstream of geneencoding CH or CL of human antibody of the vector for expression ofhumanized antibody mentioned in the above (1) to construct a vector forexpression of human chimeric antibody.

In addition, cDNA encoding VH or VL of non-human animal is amplified byPCR using a synthetic DNA having a recognition sequence of anappropriate restriction enzyme at both terminals and each of them iscloned to the vector for expression of recombinant antibody mentioned inthe above (1).

(4) Construction of cDNA Encoding V Region of Humanized Antibody

cDNAs encoding VH or VL of a humanized antibody can be obtained asfollows.

First, amino acid sequences of framework region (hereinafter referred toas “FR”) in VH or VL of a human antibody to which amino acid sequencesof CDRs in VH or VL of an antibody derived from a non-human animalantibody are transplanted are selected. Any amino acid sequences of FRin VH or VL of a human antibody can be used, so long as they are fromhuman.

Examples include amino acid sequences of FRs in VH or VL of humanantibodies registered in database such as Protein Data Bank, and aminoacid sequences common to subgroups of FRs in VH or VL of humanantibodies [Sequences of Proteins of Immunological Interest, US Dept.Health and Human Services (1991)], and the like.

In order to inhibit the binding activity of the antibody, amino acidsequences having high homology (at least 60% or more) with the aminoacid sequence of FR in VH or VL of the original antibody is selected.

Then, amino acid sequences of CDRs of VH or VL of the original antibodyare grafted to the selected amino acid sequence of FR in VH or VL of thehuman antibody, respectively, to design each amino acid sequence of VHor VL of a humanized antibody.

The designed amino acid sequences are converted to DNA sequences byconsidering the frequency of codon usage found in nucleotide sequencesof genes of antibodies [Sequence of Proteins of Immunological Interest,US Dept. Health and Human Services (1991)], and the DNA sequenceencoding the amino acid sequence of VH or VL of a humanized antibody isdesigned.

(5) Modification of Amino Acid Sequence of V Region of HumanizedAntibody

It is known that when a humanized antibody is produced by simplygrafting only CDRs in VH and VL of an antibody derived from a non-humananimal into FRs of VH and VL of a human antibody, its antigen bindingactivity is lower than that of the original antibody derived from anon-human animal [BIO/TECHNOLOGY, 9, 266 (1991)].

In humanized antibodies, among the amino acid sequences of FRs in VH andVL of a human antibody, amino acid residues which directly relate tobinding to an antigen, amino acid residues which interact with an aminoacid residue in CDR, and amino acid residues which maintain thethree-dimensional structure of an antibody and indirectly relate tobinding to an antigen arc identified and modified to amino acid residueswhich arc found in the original non-human antibody to thereby increasethe antigen binding activity which has been decreased.

In order to identify the amino acid residues relating to the antigenbinding activity in FR, the three-dimensional structure of an antibodyis constructed for analysis by X-ray crystallography [J. Mol. Biol.,112, 535 (1977)], computer-modeling [Protein Engineering, 7, 1501(1994)] or the like.

In addition, a modified humanized antibody having appropriateantigen-binding activity can be identified by repetition of variousattempts for producing several kinds of modification of each antibodyand examining the correlation between each of the modified antibodiesand its antigen binding activity.

The modification of the amino acid sequence of FR in VH and VL of ahuman antibody can be accomplished using various synthetic DNA formodification by PCR as described in (4). The nucleotide sequence of theamplified product by the PCR is determined according to the method asdescribed in (2) so as to confirm the intended modification has beenoccured.

(6) Construction of Vector for Expression of Humanized Antibody

A vector for expression of humanized antibody can be constructed bycloning each cDNA encoding VH or VL of a constructed recombinantantibody into upstream of each gene encoding CH or CL of the humanantibody in the vector for expression of recombinant antibody asdescribed in (1).

For example, recognitition sequences of appropriate restriction enzymesare introduced into the 5′-end of synthetic DNAs positioned at both endsamong synthetic DNAs used for the construction of VH or VL of thehumanized antibody in (4) and (5) to enable to clone the VH or VL in theupstream of each gene encoding CH or CL of the human antibody in theexpression vector for humanized antibody as described in the above (1),so as to express them as an appropriate form.

(7) Transient Expression of Recombinant Antibody

In order to efficiently evaluate the antigen binding activity of varioushumanized antibodies produced, the recombinant antibodies can beexpressed transiently using the vector for expression of recombinantantibody as described in (3) and (6) or the modified expression vectorthereof.

Any cell can be used as a host cell for introduction of the expressionvector, so long as the host cell can express a recombinant antibody.Generally, COS-7 cell (ATCC CRL1651) is used [Methods in Nucleic AcidsRes., CRC Press, 283 (1991)]. Examples of the method for introducing theexpression vector into COS-7 cell include a DEAE-dextran method [Methodsin Nucleic Acids Res., CRC Press, 283 (1991)], a lipofection method[Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], and the like.

(8) Establishment of Transformant which Stably Expresses RecombinantAntibody and Preparation of Recombinant Antibody

A transformant which stably expresses a recombinant antibody can beobtained by introducing the vector for expression of recombinantantibody described in (3) and (6) into an appropriate host cell.

Examples of the method for introducing the expression vector into a hostcell include electroporation [Japanese Published Unexamined PatentApplication No. 257891/90, Cytotechnology, 3, 133 (1990)] and the like.

As the host cell into which a vector for expression of a recombinantantibody is introduced, any cell can be used, so long as it is a hostcell which can express the recombinant antibody. Examples include CHO-K1(ATCC CCL-61), DUKXB11 (ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO-S(Life Technologies, Cat. No. 11619), rat YB2/3HL.P2.G11.16Ag.20 cell(also referred to as YB2/0; ATCC CRL1662), murine myeloma NSO, murinemyeloma SP2/0-Ag14 cell (ATCC CRL1581), murine P3X63-Ag8.653 cell (ATCCCRL1580), CHO cell in which a dihydrofolate reductase gene (hereinafterreferred to as “dhfr”) is deficient [Proc. Natl. Acad Sci. U.S.A., 77,4216 (1980)], lectin resistant line Lec13 [Somatic Cell and Moleculargenetics, 12, 55 (1986)], CHO cell in which α1,6-fucosyltransaferse geneis deficient (WO 2005/35586, WO 02/31140), and the like.

In addition, host cells in which activity of a protein such as an enzymerelating to synthesis of an intracellular sugar nucleotide, GDP-fucose,a protein such as an enzyme relating to the modification of a sugarchain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in a complex typeN-glycoside-linked sugar chain, or a protein relating to transport of anintracellular sugar nucleotide, GDP-fucose, to the Golgi body areintroduced is decreased or deleted, such as CHO cell in whichα1,6-fucosyltransferase gene is defected as described in WO05/35586,WO02/31140, can also be used.

After introduction of the expression vector, transformants which stablyexpress a recombinant antibody are selected by culturing in a medium foranimal cell culture containing an agent such as G418 sulfate(hereinafter referred to as “G418”) (Japanese Published UnexaminedPatent Application No. 257891/90).

Examples of the medium for animal cell culture include RPMI1640 medium(manufactured by Invitrogen), GIT medium (manufactured by NihonPharmaceutical), EX-CELL301 medium (manufactured by JRH), IMDM medium(manufactured by Invitrogen), Hybridoma-SFM medium (manufactured byInvitrogen), a derivative medium thereof containing various additivessuch as FBS.

The recombinant antibody can be produced and accumulated in a culturesupernatant by culturing the selected transformants in a medium. Theexpression amount and the antigen binding activity of the recombinantantibody in the culture supernatant can be measured by ELISA or thelike.

Also, in the transformant, the expression amount of the recombinantantibody can be increased by using DHFR amplification system or the likeaccording to the method disclosed in Japanese Published UnexaminedPatent Application No. 257891/90.

The recombinant antibody can be purified from the culture supernatant ofthe transformant by protein A column [Monoclonal Antibodies-Principlesand practice, Third edition, Academic Press (1996), Antibodies-ALaboratory Manual, Cold Spring Harbor Laboratory (1988)].

For example, the recombinant antibody can be purified by a combinationof gel filtration, ion-exchange chromatography, ultrafiltration and thelike.

The molecular weight of the H chain or the L chain of the purifiedrecombinant antibody or the antibody molecule as a whole is determinedby polyacrylamide gel electrophoresis (hereinafter referred to as“SDS-PAGE”) [Nature, 227, 680 (1970)], Western blotting [MonoclonalAntibodies-Principles and practice, Third edition, Academic Press(1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory(1988)], and the like.

3. Method of Controlling Effector Activity of Antibody

As a method for controlling an effector activity of the anti-Aβ oligomerhumanized antibody of the present invention, a method for controlling anamount of fucose (hereinafter, referred to also as “core fucose”) whichis bound in α-1,6 linkage to N-acetylglucosamine (GlcNAc) present in areducing end of a complex type N-linked sugar chain which is bound toasparagine (Asn) at position 297 of an Fc region of an antibody(WO2005/035586, WO2002/31140, and WO00/61739), a method for controllingan effector activity of a monoclonal antibody by modifying amino acidgroup(s) of an Fc region of the antibody, a method for engineering asubclass of the antibody, and the like are known. The effector activityof the anti-Aβ oligomer humanized antibody of the present invention canbe controlled by using any of the methods.

The “effector activity” means an antibody-dependent activity which isinduced via an Fc region of an antibody. As the effector activity, anantibody-dependent cellular cytotoxicity (ADCC activity), acomplement-dependent cytotoxicity (CDC activity), an antibody-dependentphagocytosis (ADP activity) by phagocytic cells such as macrophages anddendritic cells, and the like are known.

By controlling a content of core fucose of a complex type N-linked sugarchain of Fc of an antibody, an effector activity of the antibody can beincreased or decreased. As a method for reducing a content of fucosewhich is bound to a complex type N-linked sugar chain bound to Fc of theantibody, an antibody to which fucose is not bound can be obtained bythe expression of an antibody using a CHO cell which is deficient in agene encoding α1,6-fucosyltransferase. The antibody to which fucose isnot bound has a high ADCC activity.

On the other hand, according to a method for increasing a content offucose which is bound to a complex type N-linked sugar chain bound to Fcof an antibody, an antibody to which fucose is bound can be obtained bythe expression of an antibody using a host cell into which a geneencoding α1,6-fucosyltransferase is introduced. The antibody to whichfucose is bound has a lower ADCC activity than the antibody to whichfucose is not bound.

Further, by modifying amino acid residue(s) in an Fc region of anantibody, the ADCC activity or CDC activity can be increased ordecreased. For example, the CDC activity of an antibody can be increasedby using the amino acid sequence of the Fc region described inUS2007/0148165. Further, the ADCC activity or CDC activity can beincreased or decreased by modifying the amino acid as described in U.S.Pat. Nos. 6,737,056, or 7,297,775 or 7,317,091.

In addition, it is known that among human IgG subclass, the effectoractivity of IgG2 and IgG4 subclasses is lower than those of IgG1 andIgG3 subclasses. Therefore, the antibody having a lower effectoractivity can be prepared by replacing Fc region with that of theantibody subclass having a lower effector activity.

With regard to the stabilization of an antibody of IgG2 and IgG4subclasses, stable IgG2 or IgG4 antibody in which an effector activityis controlled can be prepared using a methods described inWO2006/075668, WO2006/035586 and the like.

Moreover, it is possible to obtain an antibody in which the effectoractivity of the antibody has been controlled, by using the combinationof the above-mentioned methods for one antibody.

4. Treatment Method Using Anti-AP Oligomer Antibody of the PresentInvention

It has been suggested that the decline in memory accompanying AD relatesto the synapse dysfunction caused by soluble Aβ oligomers [Klein W. L.,2001. Trends Neurosci; Selkoe D J. 2002, Science]. Accordingly, there isa possibility that excessive accumulation and deposition of Aβ oligomerstriggers a complex downstream cascade which results in AD.

In the present invention, the treatment does not necessarily haveperfect treatment effects or preventive effects on the organ and thetissue which show the symptoms due to the disorder or disease but mayprovide a part of these effects.

The treatment of AD in the present invention means improving at leastone symptom which may occur due to AD. Examples include the improvementor suppression of the cognitive dysfunction, the improvement or thesuppression of the neuritic plaque formation, the improvement or thesuppression of synapse dysfunction, and the decrease and the suppressionof Aβ accumulation in brain tissues, blood, or the like.

Here, the cognitive dysfunction includes, for example, impairedlong-term/short-term memory, impaired object recognition memory,impaired spatial memory, and impaired emotional memory.

In addition, as a therapeutic method using the anti-Aβ oligomerhumanized antibody of the present invention, examples include a methodfor suppressing the cognitive dysfunction, a method for suppressing AD,a method for suppressing the progression of AD, a method for suppressingthe neuritic plaque formation, a method for suppressing the Aβaccumulation, a method for neutralizing (suppressing) the neurotoxicityactivity, a method for inhibiting Aβ amyloid fiber formation, and amethod for neutralizing (suppressing) the synapse toxicity activity.

Furthermore, examples of other configurations include a method for atleast one of the prevention and the treatment of cognitive dysfunctionand a method for at least one of the prevention and the treatment of AD.

The therapeutic agent comprising the monoclonal antibody or antibodyfragment of the present invention or derivatives thereof may consist ofonly the antibody or antibody fragment or derivatives thereof as anactive ingredient, and is preferably supplied as a pharmaceuticalformulation produced by an appropriate method well known in thetechnical field of pharmaceutics, by mixing it with one or morepharmaceutically acceptable carriers.

Examples of a route of administration include oral administration andparenteral administration, such as buccal, tracheal, rectal,subcutaneous, intramuscular or intravenous administration.

Examples of the dosage form includes sprays, capsules, tablets, powder,granules, syrups, emulsions, suppositories, injections, ointments, tapesand the like.

The pharmaceutical preparation suitable for oral administration includesemulsions, syrups, capsules, tablets, powders, granules and the like.

Liquid preparations such as emulsions and syrups can be produced using,as additives, water; sugars such as sucrose, sorbitol and fructose;glycols such as poly,ethylene glycol and propylene glycol; oils such assesame oil, olive oil and soybean oil; antiseptics such asp-hydroxybenzoic acid esters; flavors such as strawberry flavor andpeppermint; and the like.

Capsules, tablets, powders, granules and the like can be produced using,as additives, excipients such as lactose, glucose, sucrose and mannitol;disintegrating agents such as starch and sodium alginate; lubricantssuch as magnesium stearate and talc; binders such as polyvinyl alcohol,hydroxypropylcellulose and gelatin; surfactants such as fatty acidester; plasticizers such as glycerin; and the like.

The pharmaceutical preparation suitable for parenteral administrationincludes injections, suppositories, sprays and the like.

Injections can be prepared using a carrier such as a salt solution, aglucose solution and a mixture of both thereof.

Suppositories can be prepared using a carrier such as cacao butter,hydrogenated fat or carboxylic acid.

Sprays can be prepared using the antibody or antibody fragment of thepresent invention as such or using it together with a carrier which doesnot stimulate the buccal or airway mucous membrane of the patient andcan facilitate absorption of the compound by dispersing it as fineparticles.

The carrier includes lactose, glycerol and the like. It is possible toproduce pharmaceutical preparations such as aerosols and dry powders.

In addition, the components exemplified as additives for oralpreparations can also be added to the parenteral preparations.

EXAMPLE 1 Preparation of Anti-Anβ Oligomer Humanized Antibody (1) Designof Amino Acid Sequences for VH and VL of Anti-Aβ Oligomer HumanizedAntibody 6E4

The amino acid sequence of the VH of the anti-Aβ oligomer humanizedantibody 6E4 was designed as follows.

First, the CDR sequence of the amino acid sequence (SEQ ID NO:8) wasdetermined as the VH of the anti-Aβ oligomer mouse monoclonal antibody6E4 antibody which was prepared in the reference example, based on thereport by Kabat, et al. [Sequences of Proteins of ImmunologicalInterest, US Dept. Health and Human Services (1991)]. As a result, theamino acid sequences of the CDRs 1 to 3 of the VH were set to be theones represented by SEQ ID NOs: 1 to 3.

Then, an amino acid sequence of the anti-Aβ oligomer oligomer 6E4humanized antibody was designed as follows. In order to implant theamino acid sequences (SEQ ID NOs: 1 to 3) of the CDRs 1 to 3 of the VH,the amino acid sequence of the FR of the VH in the humanized antibodywas selected.

Kabat, et al. have classified VHs of the various known human antibodiesinto subgroups (HSG I to III) based on the homologies of their aminoacid sequences, and have reported the common sequences for each of thesubgroups [Sequences of Proteins of Immunological Interest, US Dept.Health and Human Services (1991)].

Thus, the homologies between the amino acid sequences of the FRs of thecommon sequences in the subgroups I to III of the VH of the humanantibody and the amino acid sequences of the FRs of 6E4VH were searched.As a result of searching the homologies, the homologies of HSGI, HSGIIand HSGIII were 75.9%, 54.0% and 57.5%, respectively. Accordingly, theamino acid sequence of the FR of 6E4VH had the highest homology with thesubgroup I.

Based on the above result, each of the amino acid sequences (SEQ IDNOs:1 to 3) of the CDRs of 6E4VH were grafted at appropriate positionsin the amino acid sequence of the FR of the common sequence in thesubgroup I of the VH of the human antibody. Accordingly, the amino acidsequence 6E4HV0 (SEQ ID NO:12) of the VH of the anti-Aβ oligomer 6E4humanized antibody was designed.

Next, in the same manner as the H chain, the sequences of the CDRs 1 to3 of the amino acid sequence (SEQ ID NO:10) of the VL were decided asthe amino acid sequences of SEQ ID NOs:4 to 6, respectively, and theamino acid sequence of the VL of the anti-AP oligomer 6E4 humanizedantibody was designed as follows.

In order to graft the amino acid sequences (SEQ ID NOs:4 to 6) of theCDRs 1 to 3 of the VL of the 6E4 antibody, respectively, the amino acidsequence of the FR of the VL in the human antibody was selected.

Kabat, et al. have classified VLs of the various known human antibodiesinto subgroups (HSG I to IV) based on the homologies of their amino acidsequences, and have reported the common sequences for each of thesubgroups [Sequences of Proteins of Immunological Interest, US Dept.Health and Human Services (1991)].

Thus, the homologies between the amino acid sequences of the FRs of thecommon sequences in the subgroups I to IV of the VL of the humanantibody and the amino acid sequences of the FRs of 6E4VL were searched.As a result of searching the homologies, the homologies of HSGI, HSGII,HSGIII, and HSGIV were 67.5%, 86.3%, 68.8%, and 76.3%, respectively.Accordingly, the amino acid sequence of the FR of 6E4VL had the highesthomology with the subgroup II.

Based on the above result, each of the amino acid sequences (SEQ IDNOs:4 to 6) of the CDRs of 6E4VL were grafted at appropriate positionsin the amino acid sequence of the FR of the common sequence in thesubgroup II of the VL of the human antibody.

However, Leu at position 110 of the amino acid sequence (SEQ ID NO:10)of 6E4VL is not the amino acid residue which is used most frequently butthe amino acid residue which is used with relatively high frequency atthe corresponding sites in the amino acid sequences of the FRs of thehuman antibodies exemplified by Kabat, et al.

Accordingly, the amino acid residue seen in the amino acid sequence ofthe above 6E4 was employed. As described above, the amino acid sequence(SEQ ID NO:14) of the VL of the anti-Aβ oligomer 6E4 humanized antibody,6E4LV0, was designed.

The designed amino acid sequence 6E4HV0 of the VH and amino acidsequence 6E4LV0 of the VL in the anti-Aβ oligomer 6F4 humanized antibodyare the sequences constructed by grafting only the amino acid sequencesof the CDRs of an anti-Aβ oligomer mouse monoclonal antibody 6E4 to theamino acid sequence of the selected FR of the human antibody.

In general, however, in the construction of a humanized antibody, thebinding activity is reduced in many cases where the amino acid sequencesof the CDRs of the mouse antibody is simply implanted to the FR of thehuman antibody.

For this reason, in order to prevent the decrease in binding activity,the modification of the amino acid residues that is considered to affectthe binding activity among the amino acid residues that are located inthe FRs of the human antibody and different from those of the mouseantibody is carried out together with the graft of the amino acidsequences of the CDRs.

Thus, in this Example, the amino acid residues of the FR, which areconsidered to affect the binding activity, were identified in thefollowing manner.

First, the three-dimensional structure of an antibody V region(hereinafter, referred to as an HV0LV0) comprising the amino acidsequence 6E4HV0 of the VH and the amino acid sequence 6E4LV0 of the VLin the anti-Aβ oligomer 6E4 humanized antibody designed as describedabove was constructed using a computer modeling technique.

The construction of the three-dimensional structure coordinates and thedisplay of the three-dimensional structure were carried out usingDiscovery Studio (Accelrys, Inc.) in accordance with the instructionsattached thereto. The computer model of the three-dimensional structureof the V region in the anti-Aβ oligomer mouse monoclonal antibody 6E4was also constructed in the same manner.

Furthermore, the amino acid residues which were different from that in6E4 were selected among the amino acid sequences of the FR in the VIIand the VL of HV0LV0, the amino acid sequence with the modification tothe amino acid residues of 6E4 was prepared, and the three-dimensionalstructure model was constructed in the same manner.

These resultant three-dimensional structures of the V regions in 6E4,HV0LV0 and the modified forms were compared, and the amino acid residueswhich were considered to affect the binding activity of the antibodywere identified.

As a result, as the amino acid residues which are considered to changethe three-dimensional structure of the antigen binding sites and affectthe binding activity of the antibody among the amino acid residues ofthe FRs in HV0LV0, Lys at position 12, Val at position 20, Arg atposition 38, Gln at position 39, Leu at position 45, Met at position 48,Arg at position 67, Val at position 68, Ile at position 70, Ala atposition 72, Thr at position 74 and Ala at position 97 in the amino acidsequence represented by SEQ ID NO:12 in 6E4HV0 and Ile at position 2,Pro at position 15, Gln at position 50 and Tyr at position 92 in 6E4LV0were selected, respectively.

At least one amino acid sequence among these selected amino acidresidues were modified to the amino acid residue which existed at thesame site in 6E4, and thereby VH and VL of the humanized antibody whichcomprises an amino acid sequence comprising various modifications weredesigned.

Specifically, in the VH, at least one modification among the amino acidmodifications in which Lys at position 12 was substituted with Val, Valat position 20 was substituted with Leu, Arg at position 38 wassubstituted with Lys, Gln at position 39 was substituted with Leu, Leuat position 45 was substituted with Phe, Met at position 48 wassubstituted with Ile, Arg at position 67 was substituted with Lys, Valat position 68 was substituted with Ala, Ile at position 70 wassubstituted with Leu, Ala at position 72 was substituted with Val, Thrat position 74 was substituted with Lys and Ala at position 97 wassubstituted with Thr in the amino acid sequence represented by SEQ IDNO:12 was introduced.

In addition, in the VL, at least one modification was introduced amongthe amino acid modifications in which Ile at position 2 was substitutedwith Val, Pro at position 15 was substituted with Leu, Gln at position50 was substituted with Lys, and Tyr at position 92 was substituted withPhe in the amino acid sequence represented by SEQ ID NO:14.

As the antibody V region of the anti-Aβ oligomer 6E4 humanized antibodyin which at least one amino acid residue in FR of HV0LV0 was modified,HV0LV0, HV0LV2a, HV0LV2b, HV0LV2c, HV0LV2d, HV0LV3, HV0LV4, HV2LV0,HV2LV2a, HV2LV2b, HV2LV2c, HV2LV2d, HV2LV3, HV2LV4, HV3LV0, HV3LV2a,HV3LV2b, HV3LV2c, HV3LV2d, HV3LV3, HV3LV4, HV4LV0, HV4LV2a, HV4LV2b,HV4LV2c, HV4LV2d, HV4LV3, HV4LV4, HV5LV0, HV5LV2a, HV5LV2b, HV5LV2c,HV5LV2d, HV5LV3, HV5LV4, HV7aLV0, HV7aLV2a, HV7aLV2b, HV7aLV2c,HV7aLV2d, HV7aLV3, HV7aLV4, HV7bLV0, HV7bLV2a, HV7bLV2b, HV7bLV2c,HV7bLV2d, HV7bLV3, HV7bLV4, HV9LC0, HV9LV2a, HV9LV2b, HV9LV2c, HV9LV2d,HV9LV3, HV9LV4, HV12LV0, HV12LV2a, HV12LV2b, HV12LV2c, HV12LV2d, HV12LV3and HV12LV4 were designed.

FIG. 1 and FIG. 2 show the amino acid sequences of H chain variableregions HV2, HV3,HV4, HV5, HV7b, HV9 and HV12, respectively, and theamino acid sequences of L chain variable regions LV2a, LV2b, LV2c, LV2d,LV3 and LV4, respectively.

(2) Production Anti-Aβ Oligomer Humanized Antibody

The DNA encording the amino acid sequence in the variable region of theanti-Aβ oligomer humanized antibody was designed with the codons used inthe DNAs (SEQ ID NOs:7 and 9) encording the amino acid sequences of6E4VH and 6E4VL. In the amino acid modification, the DNA was designedwith the codons frequently used in mammalian cells.

The DNA sequences encording the amino acid sequences of 6E4HV0 and6E4LV0 in the anti-Aβ oligomer 6E4 humanized antibody are shown as SEQID NOs:11 and 13, respectively. In addition, the codons used in the DNAsencording the amino acid sequences of 6E4VH and 6E4VL was used fordesigning the variable region with the amino acid modification.

Using these DNA sequences, the expression vector of the humanizedantibody was constructed, and the humanized antibody was expressed.

(3) Construction of cDNA Encording VH of Anti-Aβ Oligomer HumanizedAntibody

The cDNA encording the amino acid sequence 6E4HV0 of the VH in theanti-Aβ oligomer humanized antibody shown as SEQ ID NO:12, which hadbeen designed in the above (1), and HVI2 shown in FIG. 1, which had beendesigned in the above method (2) were produced by total chemicalsynthesis.

(4) Construction of cDNA Encording VL of Anti-Aβ Oligomer HumanizedAntibody

The cDNA encording the amino acid sequence 6E4LV0 of the VL in theanti-Aβ oligomer humanized antibody shown as SEQ ID NO:14, which hadbeen designed in the above (1), and LV2a and LV4 shown in FIG. 2, whichhad been designed in the above method (2) were produced by totalchemical synthesis.

(5) Construction of Anti-Aβ Oligomer Humanized Antibody ExpressionVectors

The cDNA encording 6E4LV0 or HV12 and the cDNA encording any one of6E4LV0, LV2a and LV4 obtained in the above (3) and (4) were insertedinto appropriate positions in humanized antibody expression vectorpKANTEX93 disclosed in WO 97/10354 to construct various anti-Aβ oligomerhumanized antibody expression vectors.

(6) Expression of Anti-Aβ Oligomer Humanized Antibody using Animal Cells

The expression of the anti-Aβ oligomer humanized antibody in animalcells was carried out by a conventional method [Antibody Engineering, APractical Guide, W. H. Freeman and Company (1992)] using the anti-Aβoligomer humanized antibody expression vectors obtained in the above (5)to yield the transformant for producing the anti-Aβ oligomer humanizedantibody (HV0LV0, HV0LV2a, HV0LV4 and HV12LV4).

As an animal cell line, CHO/DG44 cell line derived by double knockout ofα1,6-fucosyltransferase (FUT8) gene (hereinafter, referred to as FUT8knockout CHO cells) was used. It has been known that no fucose is addedto the core part of the complex type N-linked sugar chain of theantibody expressed with this host cell strain (WO 2002/31140).

(7) Preparation of Purified Anti-Aβ Oligomer Humanized Antibody

After the transformant obtained in (6) of this example was cultured by aconventional culture method, the cell suspension was collected. Then,the centrifugation was carried out for 20 minutes under the condition of3000 rpm and 4° C. After collection, the culture supernatant wasfilter-sterilized with a Millex GV filter having a pore diameter of 0.22μm.

After filling 0.5 ml of Proscp vA High Capacity (manufactured byMillipore Corporation) into a column with a diameter of 0.8 cm, 5.0 mLof purified water and 5.0 mL of PBS buffer (pH7.4) were subsequentlypassed thereto to equilibrate the carrier.

Then, after the culture supernatant was passed through the column, thecolumn was washed with 5.0 mL of PBS buffer (pH 7.4). After washing, theantibody adsorbed to the carrier was eluted by 2.0 mL of 0.1 mol/Lcitrate buffer (pH 5.0), 2.0 mL of 0.1 mol/L citrate buffer (pH 3.5),and 2.0 mL of 0.1 mol/L citrate buffer (pH 3.0) in this order.

The elution was obtained by 500 μL over 4 fractions. Thereafter,SDS-PAGE analysis was performed on the thus obtained purified fractions.The fractions of which the elution of the target protein was detectedwere collected, and subjected to dialysis over a whole day and night at4° C. using 150 mmol/L of NaCl and 10 mmol/L of Na citrate solution(pH6.0).

After the dialysis, anti-Aβ oligomer humanized antibody solution wascollected and subjected to filter-sterilization using Millex GV(Millipore Corporation) with a pore diameter of 0.22 μm. The absorbanceat 280 nm (0D280 nm) was measured using an absorbance measurer (SHIMADZUUV-1700) to calculate the density of the respective purified anti-Aβoligomer humanized antibodies.

As a result, four types of anti-Aβ oligomer humanized antibodyconsisting HV0LV0 comprising 6E4HV0 as the VH and 6E4LV0 as the VL ofthe antibody, HV0LV2a comprising 6E4HV0 as the VH and LV2a as the VL ofthe antibody, HV0LV4 comprising 6E4HV0 as the VH and LV4 as the VL ofthe antibody and HV12LV4 comprising HV 12 as the VH and LV4 as the VL ofthe antibody, respectively, were produced.

EXAMPLE 2 Production of Antigen (1) Preparation of Aβ Oligomers

Amyloid β-protein, Human 1-42 peptide (manufactured by PeptideInstitute, Inc.) was dissolved at 1 mmol/L using hexafluoroisopropanol.After sonication for 10 minutes, the prepared solution was dried up inair at room temperature overnight.

Thereafter, 4 μL of dimethylsulfoxide was added thereto, andthree-minute sonication was performed. Into the prepared solution, 200μL of acetic acid solution (pH4.5) was added, and the resulting materialwas kept overnight in a stationary manner at 4° C. to produce Aβoligomers.

(2) Preparation of Aβ Monomers

Biotin-beta-Amyloid, Human 1-40 (manufactured by AnaSpec, Inc.) wasdissolved at 250 μmol/L using 0.1% of ammonia water. After the preparedsolution was subjected to sonication for five minutes, the resultant wascentrifuged under the conditions of 16,000 rpm and 4° C. for 60 minutes.Thus, Aβ monomers were prepared by collecting the supernatant.

EXAMPLE 3 Evaluation on Anti-Aβ Oligomer Humanized Antibody Activity

(1) Evaluation on Binding Activity of Anti-Aβ Oligomer HumanizedAntibody Against Aβ Oligomers using Biacore

In order to analyze the binding activity of each of anti-Aβ oligomerhumanized antibodies (HV0LV0, HV0LV2a, HV0LV4 and HV12LV4) against Aβoligomers in the chemical kinetics manner, the binding activitymeasurement was carried out using the surface plasmon resonance method(SPR method).

All the following operations were performed using Biocore T100(manufactured by GE Healthcare Bio-sciences). The Aβ oligomers obtainedin (1) of Example 2 were immobilized to the CM5 sensor chip (GEHealthcare Bio-sciences) using the amine coupling method.

Onto the chip to which the Aβ oligomers were immobilized for measurementsamples prepared to have 8 levels of density by diluting from 30 μg/mLby doubling dilution in a stepwise manner (HV0LV0, HV0LV2a, HV0LV4 andHV12LV4) was sequentially injected and measured in the order from thelower density based on the automatic program of the multi-kinetics.

Using the analysis software attached to the device, Biacore T100Evaluation software (manufactured by Biacore Life Science), analysis wascarried out with the Bivalent Analyte model to calculate the bindingrate constant ka and the dissociation rate constant kd of each of theantibody against Aβ oligomers.

The thus obtained binding rate constant ka1, the dissociation rateconstant kd1, and the dissociation constant KD (kd1/ka1) of each of theantibodies are shown in Table 1, respectively.

TABLE 1 Binding Activity of anti-Aβ oligomer humanized antibody to Aβoligomers Antibody ka (1/Ms) kd (1/s) KD (mol/L) HV0LV0 1.7 × 10⁴ 6.9 ×10⁻⁴ 4.0 × 10⁻⁸ HV0LV2a 6.4 × 10³ 3.9 × 10⁻⁴ 6.1 × 10⁻⁸ HV0LV4 7.0 × 10³5.0 × 10⁻⁴ 7.1 × 10⁻⁸ HV12LV4 2.7 × 10³ 8.1 × 10⁻⁴ 3.0 × 10⁻⁷

As shown in Table 1, all of the anti-Aβ oligomer humanized antibodies(HV0LV0, HV0LV2a, HV0LV4 and HV12LV4) exhibited binding ability to Aβoligomers at the affinity from 3×10⁴ to 4×10⁸ mol/L.

(2) Evaluation of Binding Activity of Anti-Aβ Oligomer HumanizedAntibody Against Aβ Monomers Using Biacore

In order to analyze the binding activity of the respective anti-Aβoligomer humanized antibodies (HV0LV0, HV0LV2a, HV0LV4 and HV12LV4)against Aβ monomers, the binding activity was measured in the samemanner as in (1) of this Example. In addition, the binding activity ofanti-Aβ antibody 6E10 (manufactured by CONVANCE) was measured as acontrol antibody.

The Aβ monomers obtained in (2) of Example (2) were immobilized to theSA sensor chip (manufactured by GE Healthcare Bio-sciences) by thebiotin capturing method. Onto the chip to which the Aβ monomers wereimmobilized, samples for measurement, which were prepared to have 5levels of density by diluting from 30 μg/mL by doubling dilution in astepwise manner (6E10, HV0LV0, HV0LV2a, HV0LV4 and HV12LV4), weresequentially injected and measured in the order from the lower densitybased on the automatic program of the multi-kinetics mode. Thesensorgrams are shown in FIG. 3.

As shown in FIG. 3, it has been found that all of four kinds of theanti-Aβ oligomer humanized antibodies (HV0LV0, HV0LV2a, HV0LV4 andHV12LV4) do not exhibit binding ability to Aβ monomers.

REFERENCE EXAMPLE 1. Preparation of Antigen

Aβ1-40 peptide was synthesized to produce a compound in whichfluorescent isothiocyanate of 6-carboxytetramethylrhodamine (6-TAMRA)(SIGMA) was chemically combined to the N-terminal thereof. Thepolymerization reaction of this compound was carried out with Aβ1-40peptide (Peptide Institute, Inc), and thereby a preparation (Aβ1-40oligomers) containing a large amount of oligomers was prepared.

2. Establishment of Antibody-Producing Hybridoma

The antigen prepared in the above-mentioned manner was immunized to thefootpad of a Balb-c mouse, and subsequently, six more additionalimmunizations were carried out. The hybridoma was produced by fusingwith Sp2/0-Ag14 cells with the use of the inguinal lymph node usingpolyethylene glycol 1500.

3. Dot Blot Analysis

The initial screening was performed by the dot blot analysis in which2.5 μl of Aβ1-42 (2.5 μg/dot) which had been pre-incubated for 18 hourswas immobilized on the nitrocellulose membrane. The non-specific bindingsite on the membrane was blocked with phosphate buffer solutioncontaining 5% of reduced-fat milk, 1% of BSA, and 0.05% of Tween-20, andthe non-specific binding site was incubated with culture solutionsupernatant.

The antibody bound to Aβ oligomer in the culture supernatant wasdetected by horseradish peroxidase marked goat anti-mouse F(ab′)₂(1:3000; Amersham) and depicted by a sensitive chemiluminescence (ECL)kit using LAS3000mini (Fujitsu, Tokyo, Japan). With such an operation,the anti-Aβ oligomer mouse monoclonal antibody 6E4 was established.

The above reference example was disclosed in PCT/JP 2009/52039 (WO2009/099176).

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skill in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

This application is based on U.S. provisional application No.61/232,038, filed on Aug. 7, 2009, the entire contents of which areincorporated hereinto by reference. All references cited herein areincorporated in their entirety.

Free Text in Sequence Listings:

-   SEQ ID NO:1—Description of artificial sequence; HCDR1 amino acid    sequence-   SEQ ID NO:2—Description of artificial sequence; HCDR2 amino acid    sequence-   SEQ ID NO:3—Description of artificial sequence; HCDR3 amino acid    sequence-   SEQ ID NO:4—Description of artificial sequence; LCDR1 amino acid    sequence-   SEQ ID NO:5—Description of artificial sequence; LCDR2 amino acid    sequence-   SEQ ID NO:6—Description of artificial sequence; LCDR3 amino acid    sequence-   SEQ ID NO:11—Description of artificial sequence; DNA sequence    encoding 6E4HV0-   SEQ ID NO:12—Description of artificial sequence; amino acid sequence    of 6E4HV0 variable region-   SEQ ID NO:13—Description of artificial sequence; DNA sequence    encoding 6E4LV0 variable region-   SEQ ID NO:14—Description of artificial sequence; amino acid sequence    of 6E4LV0 variable region-   SEQ ID NO:15—Description of artificial sequence; amino acid sequence    of HV 12 variable region-   SEQ ID NO:16—Description of artificial sequence; amino acid sequence    of LV2a variable region-   SEQ ID NO:17—Description of artificial sequence; amino acid sequence    of LV4 variable region

1. A humanized antibody which does not bind to an amyloid β proteinmonomer and binds to an amyloid β protein oligomer, and which comprisesthe following (a) antibody heavy chain variable region and (b) antibodylight chain variable region: (a) an antibody heavy chain variable regioncomprising the amino acid sequence represented by SEQ ID NO:12 or anamino acid sequence in which at least one modification among amino acidmodifications for substituting Lys at position 12 with Val, Val atposition 20 with Leu, Arg at position 38 with Lys, Gln at position 39with Leu, Leu at position 45 with Phe, Met at position 48 with Ile, Argat position 67 with Lys, Val at position 68 with Ala, Ile at position 70with Leu, Ala at position 72 with Val, Thr at position 74 with Lys andAla at position 97 with Thr is carried out in the amino acid sequencerepresented by SEQ ID NO:12, (b) an antibody light chain variable regioncomprising the amino acid sequence represented by SEQ ID NO:14 or anamino acid sequence in which at least one modifications among amino acidmodifications for substituting Ile at position 2 with Val, Pro atposition 15 with Leu, Gln at position 50 with Lys, and Tyr at position92 with Phe is carried out in the amino acid sequence represented by SEQID NO:14.
 2. A humanized antibody according to claim 1, wherein theantibody heavy chain variable region comprises the amino acid sequencerepresented by SEQ ID NO:12 or 15 and the antibody light chain variableregion comprises the amino acid sequence represented by SEQ ID NO:14,16, or
 17. 3. An anti-cognitive dysfunction agent, comprising thehumanized antibody according to claim 1 as an active ingredient.
 4. Amedicament for treating Alzheimer's disease, comprising the humanizedantibody according to claim 1 as an active ingredient.
 5. An agent forsuppressing formation of neuritic plaque, comprising the humanizedantibody according to claim 1 as an active ingredient.
 6. An inhibitorof formation of amyloid β amyloid fiber, comprising the humanizedantibody according to claim 1 as an active ingredient.
 7. A method forat least one of prevention and treatment of cognitive dysfunction,comprising administering the humanized antibody according to claim
 1. 8.A method for at least one of prevention and treatment of Alzheimer'sdisease, comprising administering the humanized antibody according toclaim
 1. 9. A method for suppressing progression of Alzheimer's disease,comprising administering the humanized antibody according to claim 1.